Phosphatidylinositol 3-kinase (PI3K) signaling plays an important role in the regulation of cellular lipid metabolism and non-alcoholic fatty liver disease (NAFLD). However, little is known about the role of the regulatory subunits of PI3K in lipid metabolism and NAFLD. In this study, we characterized the functional role of PIK3R3 in fasting-induced hepatic lipid metabolism. In this study, we showed that the overexpression of PIK3R3 promoted hepatic fatty acid oxidation via PIK3R3-induced expression of PPARα, thus improving the fatty liver phenotype in high-fat diet (HFD)-induced mice. By contrast, hepatic PIK3R3 knockout in normal mice led to increased hepatic TG levels. Our study also showed that PIK3R3-induced expression of PPARα was dependent on HNF4α. The novel PIK3R3-HNF4α-PPARα signaling axis plays a significant role in hepatic lipid metabolism. As the activation of PIK3R3 decreased hepatosteatosis, PIK3R3 can be considered a promising novel target for developing NAFLD and metabolic syndrome therapies.