Abstract: Objective To investigate the effect of microRNA122 (miRNA122) on cytotoxic activity of Gemcitabine on A549 cells. Methods The expression vector of miRNA122 was transfected using liposomes, and the CCK-8 was used to test the inhibition rate of Gemcitabine on non-small cell lung cancer A549 cells, then the score of IC₅₀was calculated. The effect of Gemcitabine on non-small cell lung cancer A549 cells was tested by colony-forming assay, and the apoptosis rate of A549 cells induced by Gemcitabine was tested by flow cytometry-Annexin/PI experiment. Results Transfection of miRNA122 enhanced the cytotoxic activity of Gemcitabine on A549 cells with the IC₅₀values reducing from (78.6±5.25) nmol/L to (10.26±1.18) nmol/L. Moreover, transfection of miRNA122 up-regulated the apoptosis of A549 cells induced by Gemcitabine with the apoptosis rates increasing from 26.24%±1.94% to 63.30%±3.96%. The results of RT-PCR and Western Blot assays showed that the expression level of miRNA122 was up-regulated, and its targeted genes were down-regulated via transfected miRNA122. Conclusion miRNA 122 can enhance the cytotoxic activity of Gemcitabine on A549 cells.