Abstract: To study the relationship between vitamin D₃ (VD₃) and the major histocompatibility complex (MHC) class II gene, the MHC class II antigen gene of yellow catfish was cloned by rapid-amplification of cDNA ends. The full-length cDNA of MHC class II is 1074 bp long and comprises a 78 bp 5′ untranslated region (UTR_, 259 bp 3′UTR, and a 708 bp open reading frame that encodes 236 amino acids. The deduced amino acid sequence showed the highest identity (69.5%) and the lowest identity (50.4%) with Leiocassis longirostris and Cyprinus carpio, respectively. Expression of the MHC class II antigen gene was relatively high in the small intestine, liver, and gill, but low in muscles and tail. It was nearly undetectable in the other tissues. Vitamin D₃ (0, 5000 IU/kg) was added to low phosphorus diets in the growth trial. The addition of dietary VD₃ significantly stimulated MHC class II gene expression in the small intestine and kidney. This study describes the molecular structure, tissue expression of the MHC class II antigen gene, and possible effects of dietary VD₃ on tissue expression of the MHC class II gene in yellow catfish, providing useful information for further studies of the function of this gene in yellow catfish.