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Vitamin D (VD) is a steroid hormone, of which vitamin D3 (VD3) is the main form in fish. Fish obtain VD3 majorly from diets, and VD3 has been widely used in aquatic feed as feed additive. As one of the essential nutrients, VD3 plays an indispensable role in the growth and health of fish. Due to the particularity of the fish living environment, the metabolic and physiological functions of VD3 in fish have some differences from those of mammals. This paper reviews the metabolic process of VD3 in fish, the main metabolites and vitamin D receptors, compares the differences in VD3 metabolism between fish and mammals, summarises the recommended dietary requirements for VD3 in several fish species, generalises the role of VD3 in fish growth performance, bone development, lipid metabolism, immune regulation, antioxidant and other potential physiological functions, as well as its synergistic effects with other nutrients. Future perspectives of VD3 application in aquaculture are proposed. Understanding the metabolic process and physiological function of VD3 is of great significance for extending VD3 application in the aquatic feed industry.