Gender and dietary fatty acids are involved in the regulation of lipid metabolism, disturbances of which can lead to pathologies such as metabolic syndrome or CVD. Possible interactions between these factors were investigated in male and female hamsters fed diets rich in either saturated fatty acids (‘butter’ diet) or in α-linolenic acid (‘linseed oil’ diet). Gender effect predominated over the diet effect on cholesterol (CH) metabolism; compared to males, females exhibited lower concentrations of plasma total CH (−20%, P<0·001), LDL-CH (−40%, P<0·001) and HDL-CH (−16%, P<0·001), together with higher LDL receptor (+40%) and lower HDL receptor (−60%) hepatic content. Triacylglycerol (TG) metabolism was affected by diet above all: compared to animals fed the ‘butter’ diet, those fed the ‘linseed oil’ diet exhibited lower plasma (−23%, P=0·046) and liver TG (−20%, P=0·026) concentration which may result from both an increased β-oxidation (P<0·001), without any change in PPARα mRNA, and a decreased hepatic lipogenesis (P=0·023), without increased sterol response element binding protein 1c (SREBP1c) mRNA. The response to diet was much more pronounced in males than in females, without gender effect on the transcription level of PPARα and SREBP1c. Finally, the ‘linseed oil’ diet decreased the insulin resistance index (−80%, P<0·001) with a more marked effect in males, in relation to their higher hepatic PPARγ expression (+90%, P=0·012). In conclusion, in our model, the response of either TG or CH to dietary fatty acids is modulated differently by gender. The possible relevance of these interactions to dietary practice should be taken into account in man.