Objective To investigate the role of urotoxin Indoxyl sulfate (IS) in inducing oxidative stress and fibrosis factors in HK-2 cells through organic anion transporter-3 (OAT-3). Methods Human normal renal tubular epithelial (HK-2) cells were subcultured. After cells grown with 80%~90% density, the experiment was divided into control group (Control) and IS treatment group (IS) cultured with 250 μmol of IS for 24h. At the same time, the expression of OAT-3 was further silenced with small interfering RNA (siRNA). RT-PCR and western blot was performed to analyze the mRNA and protein levels of oxidative stress (Nox-4) and fibrosis factors(CollagenI, TGF-β1, Smad-3, α-SMA). Finally, HK-2 cells were pre-treated with antioxidant N-acetylcysteine (NAC) and stimulated with 250 μmol of IS for 24 h. Results RT-PCR results showed that after HK-2 cells were stimulated with IS of 250 μ mol for 24h, IS significantly increased oxidative stress (Nox-4) and fibrosis factors(Collagen I, TGF-β1,Smad-3,α-SMA) in HK-2 cells(P<0.01). After OAT-3 silenced by siRNA in HK-2 cells, the mRNA and protein expression levels of oxidative stress and fibrosis factors induced by IS were significantly decreased (P<0.001). It is suggested that IS can induce their high expression through OAT-3. After HK-2 cells were pretreated with N-acetylcysteine (NAC) and stimulated with IS for 24h, it was found that NAC effectively inhibited IS induced mRNA and protein high expressions of Nox-4, Collagen I, TGF-β1,Smad-3, and α-SMA(p<0.01), showed that IS promoted the high expression of fibrosis factors in HK-2 cells through the high expression of oxidative stress. Conclusion IS uptaken by OAT-3 in HK-2 cells induces high expressions of oxidative stress and fibrosis factors