Recent cell culture and animal studies have suggested that expression of human apo C-III in the liver has a profound impact on the triacylglycerol (TAG)-rich VLDL production under lipid-rich conditions. The apoC-III Gln38Lys variant was identified in subjects of Mexican origin with moderate hypertriglyceridemia. We postulated that Gln38Lys (C3), being a gain-of-function mutation, promotes hepatic VLDL assembly/secretion. To test this hypothesis, we expressed C3 in McA-RH7777 cells and -null mice to contrast its effect with WT apoC-III (C3). In both model systems, C3 expression increased the secretion of VLDL-TAG (by 230%) under lipid-rich conditions. Metabolic labeling experiments with C3 cells showed an increase in de novo lipogenesis (DNL). Fasting plasma concentration of TAG, cholesterol, cholesteryl ester, and FA were increased in C3 mice as compared with C3 mice. Liver of C3 mice also displayed an increase in DNL and expression of lipogenic genes as compared with that in C3 mice. These results suggest that C3 variant is a gain-of-function mutation that can stimulate VLDL production, through enhanced DNL.