Guinea pigs and rats are both common animal models for hyperlipidemia studies. However, many recent studies have suggested that rats do not develop hypertriglyceridemia in response to cholesterol feeding. In the present work, the differences in triglyceride metabolism between guinea pigs and rats were investigated. Feeding a high-fat diet containing 0.1% cholesterol and 10% lard for 4 weeks led to a significant increase in plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and free fatty acid (FFA) in guinea pigs but not in rats. By contrast, hepatic TG levels in rats were greatly increased in response to the high-fat diet, while it remained unchanged in guinea pigs. Furthermore, the hepatic acyl CoA:diacylglycerol acyltransferase (DGAT) activity and microsomal triglyceride transfer protein (MTTP) mRNA levels in guinea pigs fed a high-fat diet were significantly higher than those in the control group, which implies an increased very-low-density lipoprotein (VLDL)-TG secretion rate in guinea pigs in response to a high-fat diet. Hepatic carnitine palmitoyltransferase-1 (CPT-1) activity and peroxisome proliferator-activated receptor-α (PPARα) mRNA levels were upregulated in guinea pigs, but not rats, fed a high-fat diet. These findings may explain the differences in plasma and hepatic TG concentrations between guinea pigs and rats. These results suggest that there are differences in triglyceride metabolism between the two species when fed high-fat diets.