In pregnancy, linoleic acid (LA), obtained from the maternal diet, is vital for fetal development. LA is metabolised into a number of fatty acid modulators that regulate the endocannabinoid system (ECS), and are key for normal metabolic homeostasis. We are currently consuming elevated levels of LA in Western societies, including during pregnancy. We investigated if elevated maternal linoleic acid is detrimental to the mother and her fetus during pregnancy and early life, and the mechanism for this. The primary aims of this study were to investigate if elevated maternal LA consumption during pregnancy alters: 1) the maternal microbiome; 2) the maternal cardiac ECS; 3) the fetal cardiac ECS at embryo (E) day 20; and 4) cardiovascular development, function and the cardiac ECS in offspring at postnatal (PN) day 40. Effects of LA exposure on an in vitro H9c2 cardiomyoblast model (viability, respiration, glycolytic metabolism) were also assessed. A secondary aim was to test whether any of the latter postnatal effects are reversed with a low LA diet. Female Wistar Kyoto rats were fed a diet high in LA (HLA - 6.21% of energy intake) or a control LA diet (LLA - 1.44% of energy) for 10 weeks prior to mating and throughout pregnancy. At E0, E10 and E20, maternal faeces were collected, and the microbiome analysed by 16S sequencing. At E20 and PN40 maternal and offspring rats were sacrificed, body and organ weights recorded, and mRNA extracted from heart tissue to assess transcription of endocannabinoid signalling targets using real time PCR. Hearts from PN40 rats were also assessed for coronary reactivity, and ventricular diastolic and systolic pressure-volume relationships. In addition, effects of elevated LA exposure on cell viability and mitochondrial respiration were assessed in isolated cardiomyocytes. The current results showed that throughout pregnancy there was an increased abundance of Cyanobacteria and Lentisphaerae, and decrease Verrucomicrobia in the microbiome of HLA mothers. Over time, HLA and LLA diets decreased the abundance of Actinobacteria and Tenericutes. There was an interaction effect for Verrucomicrobia in which pregnancy and diet interacted in influencing its abundance. In terms of effects of LA in H9c2 cardiomyoblasts, LA exposure reduced cell viability at concentrations between 300 and 1000 μM. However, there were no effects of an acute exposure to LA on mitochondrial respiration. Just prior to birth, an elevated maternal LA diet increased CB2 expression in maternal hearts whereas in her E20 offspring cardiac CB2 expression declined in both sexes. FAAH mRNA expression was unaltered by diet, and appeared higher in female vs. male hearts. There were no effects of a high maternal LA diet on mRNA expression of endocannabinoid receptor GPR18, DAGL alpha and DAGL beta in E20 offspring hearts. We further assessed cardiovascular development/function in adolescent rats. Data demonstrated that there were no changes in heart mass or heart: body weight ratios. Within the heart, no changes in resting or baseline coronary flow rate, or peak coronary reactivity were detected. However, there was a decline in coronary flow repayment during reactive hyperaemia in female hearts that was significant for the LLA maternal / offspring HLA (LHF) compared with LLA maternal/offspring (LLF) group. A trend of declining coronary flow repayment (after 30 s occlusion) was also observed in HLA maternal/offspring LLA (HLM) and HLA maternal/offspring HLA (HHM) compared to LLM and LHM hearts, though this trend did not achieve statistical significance. In terms of left ventricular diastolic mechanics, there was evidence of an increased diastolic stiffness in HHF hearts (an effect not evident in males), together with an unexpected increase in diastolic compliance in HLM/HHM hearts (counter to changes in HHF vs. other groups). Thus, there is evidence of sexually dimorphic effects of LA on coronary and diastolic function in developing hearts. Systolic pressure development was subtly modified, with a trend to reduced contractility (declining slopes of pressure-volume relationships, increased volume required to generate 100 mmHg pressure) in HL and HH diets in both sexes, though this also failed to achieve statistical significance. Molecular analysis of PN40 hearts demonstrated CB2, LepR, Notch1, NPPA and TGFB1A mRNAs were all expressed in higher levels in HLM vs. LHM groups (with NPPA also elevated vs. LLM). Expression changes in these mRNAs were not evident in females. Collectively, these changes provide further evidence of a sexually dimorphic effect of maternal diet on myocardial phenotype of offspring. In conclusion, a maternal diet high in linoleic acid alters the maternal microbiome and cardiac ECS signalling. This is associated with distinct ECS signalling changes, and alterations in cardiac metabolism and functionality at embryonic and early postnatal stages in offspring hearts. Male and female offspring demonstrated different pathophysiology, with data at these early life stages suggesting that female offspring of mothers consuming a diet high in LA may in particular exhibit altered cardiovascular makeup and function, and potentially greater risk developing cardiovascular disease later in life.