This study aimed to evaluate vitamin D (VD) levels on growth, feed utilization, enzymatic activity in lipid metabolism and mRNA expression for juvenile Orange-spotted grouper Epinephelus coioides (initial weight:7.40 ± 0.03 g) for 8 weeks. Six group fishes were fed diets containing different VD levels (0, 500, 1000, 2000, 4000, and 10,000 IU/kg, respectively). Compared to the control group, the weight gain rate and feed utilization rate showed a significant increase and then decreased (P < 0.05). The highest activities of alkaline phosphatase and superoxide dismutase were observed in the groups supplemented with 4000 and 1000 IU/kg VD, respectively (P < 0.05). With VD increased, triglyceride content gradually decreased (P < 0.05), whereas high-density lipoprotein content initially increased and then decreased. By comparison, cholesterol and low-density lipoprotein contents were not affected by VD supplementation levels (P > 0.05). VD supplementation levels significantly reduced liver fatty acid synthase activity (P < 0.05), but significantly promoted lipoprotein lipase and hepatic lipase activities (P < 0.05). As VD increased, liver VD deposition increased significantly in the 2000 IU/kg group. Fatty acid synthase (fas) mRNA expression was significantly down-regulated and then up-regulated (P < 0.05), whereas hepatic lipase (hl) mRNA expression levels exhibited the opposite trend. In summary, based on the broken-line regression analysis of WGR and dietary VD levels, the optimal dietary VD requirement was 930 IU/kg VD for juvenile Orange-spotted grouper Epinephelus coioides. A diet with a VD supplement from 1000 to 2000 IU/kg significantly improved the lipid metabolism in the liver for juvenile Orange-spotted grouper Epinephelus coioides, reducing fat deposition in the liver.