This paper aimed to investigate the effects of exercise on hepatic platelet-activating factor (PAF) metabolism in rats fed a high-fat diet. Thirty-two male Sprague-Dawley (SD) rats were divided into control group (C), high-fat diet group (H), exercise group (EC), and high-fat diet+exercise group (EH). Serum lipids, glucose, insulin and markers of hepatic injury after a 16-week dietary and/or exercise intervention (60 min/day, 6 times/week) were measured by biochemical analysis; liver lipidomic profiles were analyzed by liquid chromatograph-mass spectrometer (LC-MS). Gene and protein expression of enzymes related to PAF metabolism were determined by qPCR and Western blot respectively. The results showed that high-fat diet feeding significantly increased the levels of low-density lipoprotein-cholesterol (LDL-C) and liver injury markers including purine nucleoside phosphorylase (PNP) and malondialdehyde (MDA) in rats, which were decreased by exercise. Furthermore, high-fat diet feeding significantly increased the hepatic PAF content, which was also attenuated by exercise. In addition, although high-fat diet treatment resulted in an increase in the expression of both PAF synthetase (PAF-CPT and PLA2) and hydrolase (Lp-PLA2 and PAF-AH(II)), induction of PAF synthetase was much greater than that of PAF hydrolase. While exercise increased the expression of Lp-PLA2 and PAF-AH(II) and decreased the expression of PAF-CPT and PLA2, key PAF synthesizing enzymes. In conclusion, high-fat diet-induced increase in hepatic PAF content is mainly due to the increase of its pathological synthesis at the translational level. Exercise reduces hepatic PAF content in high-fat fed rats by increasing PAF hydrolysis and decreasing its synthesis.