For the same BMI, South Asians have a higher body fat percentage than Caucasians. There might be differences in the fatty acid (FA) handling in adipose tissue when both ethnicities are exposed to high-fat overfeeding. The objective of the present study was to investigate the molecular adaptation in relation to FA metabolism in response to overfeeding with a high-fat diet (OHFD) in South Asian and Caucasian men. Ten South Asian men (BMI 18–29 kg/m2) and ten Caucasian men (BMI 22–33 kg/m2), matched for body fat percentage, aged 20–40 years were included. A weight-maintenance diet (30 % fat, 55 % carbohydrate and 15 % protein) was given for 3 d followed by 3 d of overfeeding (150 % energy requirement) with a high-fat diet (60 % fat, 25 % carbohydrate and 15 % protein) while staying in a respiration chamber. Before and after overfeeding, abdominal subcutaneous fat biopsies were taken. Proteins were isolated, analysed and quantified for short-chain 3-hydroxyacyl-CoA dehydrogenase (HADH), carnitine palmitoyl-transferase 1α (CPT1a), adipose TAG lipase, perilipin A (PLINA), perilipin B, lipoprotein lipase and fatty acid binding protein 4 using Western blotting. OHFD decreased the HADH level (P < 0·05) in Caucasians more than in Asians (P < 0·05), but the baseline and after intervention HADH level was relatively higher in Caucasians. The level of CPT1a decreased in South Asians and increased in Caucasians (P < 0·05). PLINA did not change with diet but the level was higher in South Asians (P < 0·05). The observed differences in HADH and PLINA levels as well as in CPT1a response may be important for differences in the long-term regulation of energy (fat) metabolism in these populations.