Updated publication reference for PubMed record(s): 33440166. The health effect of dietary fat has been one of the most vexing issues in the nutrition field. Few animal studies have examined the impact of high-fat diets on lifespan by controlling energy intake. In this study, we found that compared to a normal diet, an isocaloric high-fat diet (IHF) significantly prolonged lifespan by decreasing the profiles of free fatty acids (FFAs) in serum and multiple tissues by downregulating FFAs anabolism and upregulating catabolism pathways in rats and flies. Proteomics analysis in rats identified PPRC1 as a key protein that was significantly upregulated by nearly 2-fold by IHF, and among the FFAs, only palmitic acid (PA) was robustly and negatively associated with the expression of PPRC1. Using PPRC1 transgenic RNAi/overexpression flies and in vitro experiments, we further demonstrated that IHF significantly reduced PA, which could upregulate PPRC1 through PPARG, resulting in improvements in oxidative stress and inflammation and prolonging lifespan.