African catfish (Clarias gariepinus) were fed four isonitrogenous diets (34 % crude protein), each containing one of two lipid (100 or 180 g/kg) and two L-carnitine (15 or 1000 mg/kg) levels. After 81 d of feeding, thirty-two fish (body weight 32 g) from each dietary group were randomly selected, sixteen fish were induced to a 3-h swim (speed of 1.5 body length (BL)/s), while the other sixteen fish were kept under resting condition. Fish fed 1000 mg L-carnitine accumulated 3.5 and 5 times more L-carnitine in plasma and muscle, respectively, than fish fed the 15 mg L-carnitine. Muscle L-carnitine content was significantly lower in exercised fish than in rested fish. High dietary lipid level (fish oil) led to an increase in muscle n-3 PUFA content and a decrease in SFA and MUFA content. In liver, the increase in dietary lipid level resulted in an increased levels of both n-6 and n-3 PUFA. L-carnitine supplementation significantly decreased n-3 PUFA content. Exercise decreased n-3 PUFA in both muscle and liver. Plasma lactate and lactate dehydrogenase, normally associated with increased glycolytic processes, were positively correlated with exercise and inversely correlated with dietary L-carnitine level. L-carnitine supplementation reduced significantly the RQ from 0.72 to 0.63, and an interaction between dietary L-carnitine and lipid was observed (P < 0.03). Our results indicate that an increase in fatty acids (FA) intake may promote FA oxidation, and both carnitine and exercise might influence the regulation of FA oxidation selectivity.