Recurrent stenosis is the main reason inducing the failure of urethral stricture treatment. Our previous study has found that the 316L type Cu bearing stainless steel (316L-Cu SS) showed antimicrobial activity and anti-encrustation performance when it was used for relieving urethral obstructer. However, whether it can reduce the occurrence of fibrosis or not, we need further investigation to compare the cellular and molecular responses of human urethral scar fibroblast cells (USFCs) on 316L-Cu SS and medical grade 316L stainless (316L SS, as a control). [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)- 2H-tetrazolium (MTS) and Transwell were used to assess the cellular responses, which confirmed that 316L-Cu SS could inhibit proliferation and migration of USFCs. Molecular expressions of fibrosis were evaluated by western blot, real-time quantitative polymerase chain reaction (qPCR), and Cu/Zn superoxide dismutase (CuZnSOD) measurement. The results indicated that up-regulating of CuZnSOD attenuated the transforming growth factor-β1 expression and phosphorylation of Smad3 after exposure to 316L-Cu SS. Besides, the content of collagen type I (COL1) and collagen type III (COL3) secreting into the culture medium measured by enzyme-linked immunosorbent assay were in accord with the results of messenger ribonucleic acids. Both of them exhibited lower levels of COL1/COL3 exposure to 316L-Cu SS, demonstrating the inhibitory performance of 316L-Cu SS against fibrosis. ? 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2019-2028, 2018.