Introduction: Bacterial infection and bile flow retardation form a vicious cycle which promotes stone formation and recurrence, and it seems that mucin overexpression plays an important role in this process. However, the mechanism of increased mucus secretion in the biliary tract by bacterial infection and its treatment remain unclear. Material and methods: Human biliary epithelial cells were induced by neutrophil elastase (NE), and H2O2 production in the cell supernatants was detected by a specific kit, and then cells were pretreated with a H2O2 inhibitor, and expression of MUC5AC was detected by real-time polymerase chain reaction (PCR), Western blot, and immunohistochemistry. Moreover, selective PKC and Nox inhibitors, apocynin and bisindolylmaleimide I, were used to pretreat cells and detect H2O2, MUC5AC mRNA and protein expression. Then, we pretreated cells with selective inhibitors or NE, and detected transforming growth factor alpha (TGF-alpha) using an ELISA kit. Results: H2O2 production increased in an NE dose-dependent manner (p < 0.001), and NE upregulated MUC5AC expression at both mRNA and protein levels, while DMTU, could reduce this high expression (p < 0.01 at mRNA level, p < 0.001 at grey analysis for western blot and p < 0.01 at mean density for immunohistochemical staining at protein level). Moreover, apocynin and bisindolylmaleimide I could reduce the H2O2 production stimulated by NE (p < 0.05), and reduce MUC5AC high expression (p < 0.01 at mRNA level, p < 0.001 at both grey analysis for western blot and mean density for immunohistochemical staining at protein level). In addition, NE induced TGF-alpha production, and any of the three selective inhibitors could reduce it (p < 0.05). Conclusions: NE-induced reactive oxygen species participated in the up regulation of MUC5AC production. Moreover, protein kinase C and NADPH oxidase (Nox) regulate MUC5AC production in NE-challenged human biliary epithelial cells.