Oxytetracycline (OTC) and Cu are prevalent in aquatic ecosystems and their pollution are issues of serious concern. The present working hypothesis is that the toxicity of Cu and OTC mixture on physiological activity of fish was different from single OTC and Cu alone. The present study indicated that, compared to single OTC or Cu alone, Cu+OTC mixture reduced growth performance and feed utilization of grass carp, escalated the contents of Cu, OTC and TG, increased lipogenesis, induced oxidative stress, damaged the mitochondrial structure and functions and inhibited the lipolysis in the liver tissues and hepatocytes of grass carp. Cu+OTC co-treatment significantly increased the mRNA abundances and protein expression of Nrf2. Moreover, we found that Cu+OTC mixture-induced oxidative stress promoted Nrf2 recruitment to the SREBP-1 promoter and increased SREBP-1-mediated lipogenesis; Nrf2 sited at the crossroads of oxidative stress and lipid metabolism, and mediated the regulation of oxidative stress and lipid metabolism. Our findings clearly indicated that OTC and Cu mixture differed in environmental risks from single antibiotic or metal element itself, and thus posed different toxicological responses to aquatic animals. Moreover, our findings suggested that Nrf2 functioned as an important antioxidant regulator linking oxidative stress to lipogenic metabolism, and thus elucidated a novel regulatory mechanism for lipid metabolism. Display omitted •Cu+OTC escalated TG content by up-regulating lipogenesis and inhibiting lipolysis.•Cu+OTC induced oxidative stress, damaged mitochondrial structure and functions.•Cu+OTC increased Nrf2 expression and promoted Nrf2 recruitment to SREBP-1 promoter.•Cu+OTC increased TG content via Nrf2-SREBP-1 pathway activated by oxidative stress.•Nrf2 sited at crossroad of oxidative stress and lipid metabolism induced by Cu+OTC.