Objective To explore the mechanism of gefitinib in the treatment of non-small cell lung cancer (NSCLC) cell lines in vitro. Methods The effects of the gefitinib in five human NSCLC cell lines (A549, SPC-A-1, H460, H1229, 95D) were studied. The inhibition of cell proliferation in each group were measured by CCK8;The status of apoptosis cells were observed using flow cytometry after PI marked;invasion of lung cancer cell inhibited by gefitinib were assessed by transwell technique;The drug was detected by western blot on the proliferation-related signaling protein. Results The proliferation and invasive capacity of NSCLC cells were inhibited by gefitinib (P < 0.05). In gefitinib group, the apoptosis rates of A549 [(9.6±0.73) %]and SPC-A-1[(14.3±1.12) %]were higher than that of control group[(3.1±0.29) %](t =11.16,P =0.001;t =4.726, P =0.009). Expression of p-AKT, p-EGFR, p-MAPK protein levels were significantly down regulated in A549 cells when gefitinib was given after 72 hours (t =6.656, P =0.003;t =16.441, P =0.0001;t =3.736, P =0.020). Conclusion Gefitinib can inhibit the proliferation and invasion of lung cancer cell, also can induce apoptosis in vitro and most likely to contribute to the inhibition of key enzymes in EGFR signaling transduction pathway.