MAD2B, an anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase zeta, is indispensible for mitotic checkpoint control and DNA repair. Previously we established MAD2B is expressed in glomerular and tubulointerstitial compartments and participates in high glucose induced podocyte injury. But its role in other renal diseases remains elusive. In the present study we aim to illustrate the potential role of MAD2B in the pathogenesis of renal fibrosis. By immunofluorescence and western blot, we found MAD2B expression is obviously increased in tubulointerstitial fibrosis (TIF) patients and unilateral ureteral obstruction (UUO) mice. It is widely accepted that resident fibroblasts are the major source of collagen-producing myofibroblasts during TIF. Therefore, we evaluated the level of MAD2B in fibroblasts (NRK-49F) exposed to TGF-β1 by immunoblotting and revealed that MAD2B is upregulated in a time dependent manner. Intriguingly, SnoN, a transcriptional repressor of TGF-β1/Smad signaling pathway, is decreased in TGF-β1 treated fibroblasts as well as the kidney cortex from TIF patients and UUO mice. Either in vitro or in vivo locally genetic depletion of MAD2B by Lentiviral transfection could preserve SnoN abundance and suppress Smad3 phosphorylation which finally dampens the fibroblast activation, ECM accumulation and alleviates the severity of TIF. However ubiquitin ligase APC/C is not involved in MAD2B mediated SnoN decline, although this process is ubiquitination dependent. In conclusion, our observation proposes that besides cell cycle management MAD2B has a profibrotic role during fibroblast activation and TIF by suppressing SnoN expression. Targeting MAD2B-SnoN pathway is a promising intervention for TIF.