We compared the efficacy of alpha-linolenic acid (alpha-LNA, n-3) and linoleic acid (LA, n-6) on orotic acid (OA)-induced fatty liver in Sprague-Dawley rats. Rats were fed semi-synthetic diets containing either LA or alpha-LNA with or without 1% OA for 2 wk. OA supplementation lowered serum lipids in LA+OA groups. In addition to the decline of serum lipids in alpha-LNA groups compared to LA groups, a further decrease was found in alpha-LNA+OA groups compared to LA+OA groups. OA-containing diets significantly increased the liver weights and triacylglycerol (TG) accumulations compared with the OA-free diets. These results were attributed to the significant increases in the activities of phosphatidate phosphohydrolase (PAP), a rate-limiting enzyme of TG synthesis, and glucose-6-phosphate dehydrogenase, a fatty acid synthesis-related enzyme. However, the increase of PAP activity was significantly less in the alpha-LNA+OA group as compared with the LA+OA group. These results suggest that dietary alpha-LNA alleviates OA-induced hepatic TG accumulation through the attenuation of hepatic TG synthesis in rats.