Abstract: In order to investigate the mechanism about the regulation of immune response by miRNAs mediating feed vitamin D₃ in the JAK-STAT pathway of yellow catfish (Pelteobagrus fulvidraco), three diets with vitamin D₃ content at 1120, 3950 and 16600 IU/kg were designed for juvenile Pelteobagrus fulvidraco. The growth trial was carried out over a 12-week period, following by an Edwardsiella ictaluri challenge experiment. Illumina high-throughput sequencing of head kidney and spleen after bacteria challenge identified differentially expressed miRNAs. The target genes of miR-194a was enriched in the JAK-STAT pathway. The relationship between miR-194a and the possible target genes jak2a and tyk2 had been verified by a dual luciferase reporter gene assay, which indicated the inhibitory role of miR-194a on jak2a and tyk2. In vitro, miR-194a was also found to respond with different concentrations of vitamin D₃, inhibiting the expression of the target genes of jak2a and tyk2. With the activation of the JAK-STAT signaling pathway, the expression of anti-inflammatory response (AIR) factors and the target genes were also activated, the NF-κB pathway was inhibited, with significant decrease in expression of pro-inflammatory factors IL-1β, IL-6 and TNF-α and the transformation of macrophages from M1-type to M2-type polarization was accelerated with the increasing of vitamin D₃ concentration (P<0.05). In conclusion, the results of this study demonstrated that miR-194a could respond to different concentration of vitamin D₃ in vivo and in vitro experiments. Dietary vitamin D₃ up-regulated the expression of miR-194a, which negatively regulated the target genes of jak2a and tyk2, and exerted indirect effects towards the downstream genes in the JAK-STAT pathway, illuminating the mechanism of miR-194a-mediated regulation of immune response by dietary vitamin D₃ through the JAK-STAT signaling pathway in Pelteobagrus fulvidraco.