Mechanisms linking pre-pregnancy obesity to increased preterm birth risk are unclear. Here, we examined the impact of pre-pregnancy obesity on metabolites, Fms-related tyrosine kinase 3 ligand (Flt3L), and proinflammatory cytokine profiles in preterm birth. We used cytokine bead array, ELISA and Gas Chromatography-Mass Spectrometry (GC-MS) to determine cytokine and metabolite profiles in maternal and cord blood samples from 124 pregnant women in Australia, who gave birth at term (n = 86) or preterm (n = 38). Besides the expected variations in birth weight and gestational age, all demographic characteristics, including pre-pregnancy body mass index, were similar between the term and preterm birth groups. Mothers in the preterm birth group had reduced Flt3L (P = 0.002) and elevated IL-6 (P = 0.002) compared with term birthing mothers. Among mothers who gave birth preterm, those with pre-pregnancy obesity had lower Flt3L levels (P = 0.02) compared with lean mothers. Flt3L and IL-6 were similar in cord blood across both groups, but TNFα levels (P = 0.02) were reduced in preterm newborns. Metabolomic analysis revealed significant shifts in essential metabolites in women with pre-pregnancy obesity, some of which were linked to preterm births. Our findings suggest that maternal pre-pregnancy obesity alters the metabolome and reduces Flt3L expression, potentially increasing risk of preterm birth.