The M2 ion channel protein is an important target against influenza A virus. In this study, H5N1 influenza A virus M2 ion channel (H5M2) gene was cloned into pcDNA4 vector. The HEK293 stable cell line expressing H5M2 was successfully established. The expression of H5M2 ion channel protein was induced only by tetracycline and confirmed by imuunofluorescence and Western blot. The ion channel activity of H5M2 was confirmed by whole cell patch-clamp recording. Fifty micromol per liter amantadine blocked the H5M2 channel conductance completely in HEK293 cells. This stable cell line may provide a model for screening inhibitors of M2 ion channel.