The study was carried out to investigate the effects on possible gene regulation in intestine and kidney of yellow catfish (Pelteobagrus fulvidraco) under the treatments of different dietary vitamin D₃ levels (0(VD0), 1243(VD2), 22 700(VD20) IU/Kg) by RNA-seq technique. 83 265 unigenes were selected from the 305 568 982 raw reads. The average length was 845.38 nt and N50 was 1620 nt. 29 224 genes were annotated by using Blast software. According to the results of gene ontology functional enrichment analysis, significant differently expressed genes Were mainly distributed on the pathways like cellular process, metabolic pathways and biological regulation. The results of KEGG pathway enrichment analysis showed that 1532 genes were involved in metabolic pathway, in which the affected genes were the most. Dietary vitamin D treatment significantly affected the genes expression. When compared with the control group (VD0 vs VD2/VD0 vs VD20), 380 genes were up-and down-regulated in common, in which 266 genes were up-regulated and 114 genes were down——regulated. Among three levels (VD0 vs VD2 vs VD20), 21 genes were up-and down-regulated in common, including 4 genes up regulated and 17 down-regulated. The results indicated that different dietary vitamin D₃ levels significantly affected the gene expressions in intestine and kidney of yellow catfish. The information of the differentially expressed genes provided evidences about the growth, metabolism and immunological response of yellow catfish.