The essential fatty acids, omega-3 and omega-6, consumed during pregnancy can benefit maternal and offspring health. For instance, they could activate a network of genes related to the nuclear receptor peroxisome proliferator-activated receptor α (Ppara) and sterol regulatory element binding transcription factor 1 (Srebf1), which play a role in fatty acid oxidation and lipogenesis. The present study aimed to investigate the effects of diets with different omega-3/omega-6 ratio consumed over three generations on blood biochemical parameters and hepatic expression of Ppara- and Srebf1-related genes. During three consecutive generations adult Wistar rats were evaluated in the postpartum period (21 days after parturition). Regardless of prenatal dietary omega-3/omega-6 ratio, an upregulation in liver tissue was observed for Rxra, Lxra and Srebf1 and a downregulation for Fasn in all the evaluated generations. The diet with higher omega-3/omega-6 ratio decreased triacylglycerol serum levels and resulted in a constant non-esterified fatty acid level. Our results indicated that the PUFAs effect on the modulation of genes related to fatty acid oxidation and lipogenesis is cumulative through generations.