〔Abstract〕 To investigate the impact of aberrant SKI expression and its mutations on the biological char- acteristics of cholangiocarcinoma cell lines QBC939 and RBE，and to explore the underlying molecular mecha- nisms. Methods The Gene Expression Profiling Interactive Analysis 2（GEPIA2）database was utilized to analyze SKI expression and its clinical relevance in cholangiocarcinoma patients. Lentiviral transduction was employed to establish QBC939 and RBE cell lines with stable SKI overexpression，mutation，or knockdown. Cell proliferation was assessed using CCK-8，colony formation，and EdU assays. Apoptosis and cell cycle distribution were analyzed by flow cytometry. Cell migration was evaluated using Transwell and wound healing assays. The effect of SKI over-expression，mutation，or knockdown on key proteins（SMAD2，SMAD3，SMAD4）in the transforming growth fac- tor-β（TGF-β)/Small mothers against decapentaplegic（SMAD）signaling pathway was examined by Western blot. Results Compared to SKI overexpression alone，the introduction of SKI mutations significantly promoted S-phase progression，enhanced proliferation and migration，and inhibited apoptosis. Mechanistically，SKI mutations sup- pressed the phosphorylation of SMAD2 and SMAD3 proteins，thereby inhibiting the transcriptional activity of the TGF-β signaling pathway. Conversely，SKI knockedown produced the opposite effects. Conclusion SKI gene mu- tation acts as a gain-of-function genetic alteration，exerting an oncogenic role in cholangiocarcinoma cells. The pri- mary mechanism involves the inhibition of the TGF-β/SMAD signaling pathway，which in turn promotes prolifera- tion and cell cycle progression，and suppresses apoptosis in QBC939 and RBE cells，ultimately driving tumor pro- gression.