The fenofibrate functions in mammals could be affected by many factors such as dietary nutrient levels and physiological status. However, this phenomenon has not been well studied in fish. The goal of our study was to investigate the effect of dietary protein contents on metabolic regulation of fenofibrate in Nile tilapia. An 8-week experiment was conducted to feed fish with four diets at two protein levels (28% and 38%) with or without the supplementation of fenofibrate (200 mg/kg body weight/day). After the trial, the body morphometric parameters, plasma biochemical parameters and qPCR data were examined. These results showed that fenofibrate significantly reduced the feeding intake and weight gain rate, increased the oxidative stress (increased plasma methane dicarboxylic aldehyde, MDA) and liver/body ratio (increased hepatosomatic index, HSI) in the LP (low protein)-fed fish. In contrast, fenofibrate exhibited a lipid-lowering (reduced hepatic lipid) effect and up-regulated the expressions of the genes related to lipid catabolism, transport and anabolic metabolism in the HP (high protein) fed-fish. The present study suggested that lipid-lowering effect of fenofibrate would be strengthened in the fish fed with HP diet containing high energy, but in the fish fed with the LP diet containing low energy, the fenofibrate treatment would cause adverse effects for metabolism. Taking together, our study showed that the metabolic regulation of fenofibrate in Nile tilapia was not only dependent on feed energy content, but also dependent on dietary nutrient composition, such as dietary protein and/or lipid levels.