The mechanistic evidence to support the cardioprotective effects of polyunsaturated fatty acids (PUFA) are controversial. The aim was to test cardioprotective mechanisms induced by PUFA supplementation against cardiac ischemia-reperfusion (IR) injury. Ten-week-old male Wistar rats (225 ± 14 g, n = 14) were divided in two groups: rats without supplementation (n = 7) and a PUFA group, supplemented by PUFA (0.6 g/kg/day; DHA:EPA = 3:1) for eight weeks (n = 7). Hearts were perfused with Krebs–Henseleit buffer for 20 min (control conditions); others were subjected to control conditions, 30 min of global ischemia and 120 min of reperfusion (IR group). Infarct size (IS) and left ventricular developed pressure (LVDP) were measured at 120 min of reperfusion. Oxidative stress biomarkers (TBARS, total carbonyls), antioxidant status (CAT, catalase; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase activity and GSH/GSSG ratio), myeloperoxidase activity, ATP levels and nuclear transcription factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappaB (NF-κB) were determined in both experimental conditions. At the end of reperfusion, hearts supplemented with PUFA showed lower IS and a higher LVDP compared with the nonsupplemented rats. Hearts in the group supplemented with PUFA showed lower levels of oxidative stress markers and higher antioxidant activity, decreased MPO activity and NF-κB and Nrf2 activation compared with the nonsupplemented group. Cardioprotective effects of PUFA are exerted through induction of anti-inflammatory and antioxidant mechanism at tissue level.