Abstract: This experiment was conducted to study the effects of dietary vitamin D₃ levels on the growth performance and the expression of TLR18, TLR19 and TLR21 in Pelteobagrus fulvidraco. A total of 800 healthy Pelteobagrus fulvidraco with an average body weight of (5.0±0.2) g were randomly assigned to 5 groups with 4 replicates per group and 40 fishes per replicate. Juvenile yellow catfish were fed with the basal diet supplemented with 1120, 2260, 3950, 8030 and 16600 IU/kg vitamin D₃. After a 12-week growth trial, 50 fish from each treatment were injected with Edwardsiella ictaluri, and the fish were challenged for 72h. The spleen, head kidney, liver and anterior intestine from fish in each group were collected at 0 and 72h after the Edwardsiella ictaluri challenge and were used to detect the expression of three toll-like receptors, i.e., TLR18, TLR19 and TLR21. Meanwhile, the muscles, head kidney, kidney, skin, brain, gill, spleen, stomach, anterior intestine and liver were collected from fresh nonexperimental yellow catfish to detect the tissue distribution of TLR18, TLR19 and TLR21. The results showed that different levels of dietary vitamin D₃ can significantly affect growth performance. The TLR18, TLR19 and TLR21 genes were expressed in all tested tissues, but the highest expression was found in the spleen. The Edwardsiella ictaluri challenge significantly increased the expression of TLR18, TLR19 and TLR21. Different levels of dietary vitamin D₃ had a significant influence on TLR18, TLR19 and TLR21 gene mRNA expression in the head kidney, spleen, liver and anterior intestine both before and after the pathogen challenge. These results showed that appropriate supplementation with dietary vitamin D₃ can stimulate the growth and pathogen resistance of juvenile yellow catfish.