The effect of activin A on tubulointerstitial fibrosis in diabetic nephropathy (DN) using streptozotocin (STZ)-induced diabetic rats and high glucose-cultured HK-2 cells was investigated. Male Wistar rats were randomized into a normal control group (NC) and diabetes mellitus group (DM). Diabetes was induced by i.p. injection of STZ. Six rats were respectively killed 4, 8, 12 and 16 weeks after model establishment in each group. The changes of kidney weight/bodyweight (KW/BW), urine albumin excretion rate (AER) and creatinine clearance rate (Ccr) were determined. The morphology of tubulointerstitium was observed by light microscopy. Further biochemical analysis was provided using immunohistochemistry and real-time polymerase chain reaction. The different parameters in high glucose-cultured HK-2 cells were monitored by western blotting or enzyme-linked immunosorbent assay (ELISA) and the intervention of rh-follistatin on them was investigated. Compared with the NC group, there was marked enlargement in the levels of KW/BW, AER, Ccr and interstitial fibrosis index, and the production of P-Smad2/3 and fibronectin in the DM group from 8 to 16 weeks. Activin betaA, mainly located in tubular epithelial cells, was significantly higher in the DM group than that in the NC group throughout the study periods. Follistatin was abundant in the NC group, but was diminished gradually in the DM group. High glucose may facilitate the synthesis of activin betaA, transforming growth factor (TGF)-beta, P-Smad2/3 and fibronectin in HK-2 cells while rh-follistatin inhibited them except TGF-beta. Activin A is involved in tubulointerstitial fibrosis in DN by inducing the production of fibronectin through Smad signal pathway.