Recent studies have reported that upregulation of disulfide-bond A oxidoreductase-like protein (DsbA-L) prevented lipid-induced renal injury in diabetic nephropathy (DN). However, the role and regulation of proximal tubular DsbA-L for renal tubulointerstitial fibrosis (TIF) remains unclear. In current study, we found that a proximal tubules-specific DsbA-L knockout mouse (PT-DsbA-L-KO) attenuated UUO-induced TIF, renal cell apoptosis and inflammation. Mechanistically, the DsbA-L interacted with Hsp90 in mitochondria of BUMPT cells which activated the signaling of Smad3 and p53 to produce connective tissue growth factor (CTGF) and then resulted in accumulation of ECM of BUMPT cells and mouse kidney fibroblasts. In addition, the progression of TIF caused by UUO, ischemic/reperfusion (I/R), aristolochic acid, and repeated acute low-dose cisplatin was also alleviated in PT-DsbA-L-KO mice via the activation of Hsp90 /Smad3 and p53/CTGF axis. Finally, the above molecular changes were verified in the kidney biopsies from patients with obstructive nephropathy (Ob). Together, these results suggest that DsbA-L in proximal tubular cells promotes TIF via activation of the Hsp90 /Smad3 and p53/CTGF axis.