Abstract: Objective To investigate the effects of krüppel-like factor 5 (KLF5) overexpression on gemcitabine (GEME)-induced lung adenocarcinoma cell apoptosis and its molecular mechanisms. Methods Lung adenocarcinoma cell line H441 were transfected with KLF5 plasmid or control plasmid. 68 h later, cells were treated with 100 nmol/L GEME for 4 h，then cell number counting and flow cytometry were applied to detect cell proliferation and apoptosis; Western blot were used to analyse the expression level of KLF5, RT-PCR were used to analyse the KLF5gene, apoptosis-related genes CD95 and BAX; Immunofluorescence staining was performed to detect the expression level of apoptosis-related protein Caspase 3. Results The overexpressions of KLF5 protein were detected in cultured- lung adenocarcinoma cell line H441 cells when KLF5 plasmids were transfected 68 h. Further flow cytometry, overexpression of KLF5 in H441 cell line affected the biological process of apoptosis significantly. No significant changes of apoptosis and expression level of CD95 and BAXin H441 cells were observed by KLF5 overexpression without GEME treatment (P >0.05). Under GEME induction, the proportion of apoptotic cells and expression level of CD95 and BAXwere increased significantly in H441 cells by KLF5 overexpression, compared with that of control (P <0.05); The overexpression of KLF5 restrained the proliferation of H441 cells; Immunofluorescence staining of Caspase 3 was also enhanced after KLF5 overexpression. Conclusion Under GEME induction, the overexpression of KLF5 promoted the apoptosis of lung adenocarcinoma cell line H441 in vitro, possibly through the inhibition of cell proliferation and repair/activation of apoptosis pathway proteins, such as Caspase 3, CD95 and BAX.