Objective To determine the inhibitory effect of a synthetic hexa-peptide GGWSHW (G peptide) derived from thrombospondin-1(TSP1) on TGF-β activation induced by angiotensin Ⅱ (AngⅡ) in cultured human renal tubular epithelial cells. Methods Human proximal tubular epithelial cell line(HK-2) was cultured in vitro， untreated HK-2 cells were acted as normal control group. HK-2 cells were then stimulated by AngⅡ for 1~24 hours (AngⅡ stimulation group)， so that optimal dosage and duration could be chosen. One hour prior to induction， HK-2 cells were pretreated with 10 μmol/L peptide G(G peptide treated group)or losartan (losartan treated group)， the blocker of Ⅰ type receptor of AngⅡ was acted as inhibitory control. The mRNA and protein levels of TSP1， TGF-β1， FN and PAI-1 were measured by RT-PCR and Western blot. Confocal microscopy and flow cytometry were performed to detect the presence of TSP1， TGF-β1 and co-positive expression of two protein， respectively. The concentrations of total and active TGF-β1 as well as FN and PAI-1 in cell culture supernatants were measured by ELISA. Additionally， the expression of Smad2 and p-Smad2 was also examined for the bioactivity of TGF-β1 signaling protein.Results AngⅡ enhanced the expression of TSP1 and TGF-β1 in a temporal-spatial dependent manner. The optimal dosage and duration were 1 μmol/L and 6 hours， for TSP1， and 0.1 μmol/L and 12 hours for TGF-β1 respectively. Comparing with untreated HK-2，the co-expression of TSP1 and TGF-β1 induced by AⅡ showed a increase of 5.4 folds. In addition， the protein level of p-Smad2 was elevated remarkedly， the mRNA level of FN and PAI-1 was up-regulated by 3 and 1.5 folds， and the concentration was increased by 2.0 and 1.9 folds respectively. Peptide G had less effect on the expression of TSP1 and TGF-β1， whereas it significantly reduced the secretion of active TGF-β1， though total level of TGF-β1 remained up-regulated. Furthermore， comparing with losartan treated group， p-Smad2 expression was reduced by 28.9%， the mRNA level of FN and PAI-1 was decreased by 34.5% and 26% respectively， and the protein levels were reduced by 11.0% and 8.9% respectively. Conclusion The inhibitory peptide derived from TSP1 effectively suppresses TGF-β1 activation through a competitive mechanism and also reduces the secretion of FN and PAI-1 associated with fibrosis.