Abstract: As a key class of immune cells, macrophages are not only the first line of defense against pathogen invasion, but also play different functions through polarization. Macrophages can secrete pro-inflammatory factors and cause excessive inflammatory response, resulting in a variety of inflammatory diseases when polarizing into M1 type. Macrophages can secrete anti-inflammatory factors and play an anti-inflammatory role when polarizing into M2 type. Macrophages are characterized by phenotypic heterogeneity and functional diversity, and it is important to target macrophage-type polarization through nutritional regulation. Vitamin D₃ has been proved to play important role in fish anti-inflammatory response. In this study, the effects of 200 mol/L vitamin D₃ on polarization phenotype of head kidney macrophages of Pelteobagrus fulvidraco were studied. The primary macrophage cells of Pelteobagrus fulvidraco head kidney were isolated and cultured. After being challenged by LPS and cAMP to induce macrophage polarization, the cell morphological changes were observed by inverted light microscopy, and the functional changes were studied by measuring survival rate, phagocytosis, reactive oxygen species and nitric oxide production, superoxide anion radical and arginase activity, as well as the related gene expression charactering in different macrophages polarization states. The results showed that vitamin D₃ reduced the mortality of M1 and M2 macrophages and enhanced the phagocytic activity of macrophages. In M1 macrophages, vitamin D₃ inhibited the production of reactive oxygen species (ROS) and inflammatory mediator nitric oxide (NO), reduced the activity of superoxide anion free radical, and decreased the expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) (P<0.05). The activity of arginase as well as the expression of interleukin-10 (IL-10) and transforming growth factor (TGF-β) in M2 cells was up-regulated by vitamin D₃ (P<0.05). In conclusion, vitamin D₃ inhibited the polarization of macrophages to M1 phenotype, promoted the polarization of macrophages to M2 phenotype, and played an anti-inflammatory role. In the current study, Nos-2 and arg-2 were found to be biomarker genes of M1 and M2 macrophages, respectively. These results preliminarily reveal the mechanism of vitamin D₃ on the polarization of head kidney macrophages of Pelteobagrus fulvidraco, and favor the regulation of macrophage phenotypes in the treatment of inflammation, which provides useful information for further study on the polarization of fish macrophages and the effect of vitamin D₃ on polarization regulation.