The over-activation of Toll-like receptors (TLRs) is a typical immune response to injury. Previous work has suggested that controlling the over-activation of TLR4-MyD88-NF-κB may represent a new therapeutic option for diabetic kidney disease (DKD). 1,25(OH)D has also been shown to exert a protective effect on DKD, although the mechanism involved has yet to be elucidated. The aim of this study was to investigate whether 1,25(OH)D protects against DKD by down-regulating the innate immune TLR-NF-κB pathway. NRK-52E cells were cultured under normal or high-glucose conditions. We then used siRNA to knock down TLR4 expression under high-glucose conditions. NRK-52E cells cultured under high-glucose conditions, and streptozotocin (STZ)-induced diabetic rats, were treated with different doses of 1,25(OH)D and used as in vitro and in vivo models, respectively. Renal biochemical indicators were then measured to evaluate the influence of 1,25(OH)D treatment on DKD in diabetic rats. Histological analysis was also performed to determine the extent of infiltration by inflammatory cells and tubulointerstitial fibrosis. Using RT-qPCR, western blotting, immunohistochemistry and immunofluorescence, we determined the expression levels of TLR4, MyD88, NF-κB p65, MCP-1 and α-SMA to investigate whether 1,25(OH)D could reduce the development of tubulointerstitial fibrosis. Knocking down TLR4 abolished the tubulointerstitial fibrosis caused by high-glucose conditions. High doses of 1,25(OH)D consistently reduced the expression of TLR4-MyD88-NF-κB in NRK-52E cells. Moreover, high doses of 1,25(OH)D had an obvious protective effect on kidney injury and inhibited the infiltration of inflammatory cells and tubulointerstitial fibrosis in diabetic rats. In conclusion, high doses of 1,25(OH)D protected against tubulointerstitial fibrosis both in vitro and in vivo by downregulating the expression of TLR4-MyD88-NF-κB.