Gefitinib is one of commonly used first-line treatment options for patients with positive EGFR mutation in non-small cell lung cancer (NSCLC). However, most patients with gefitinib treatment relapse over time due to the loss of drug sensitivity. Compound Kushen injection (CKI) has been used to treat lung cancer, including EGFR-mutated NSCLC. In this report, we examined the anti-cancer and drug sensitivity increased activities of CKI in gefitinib less sensitive NSCLC cell lines H1650 and H1975. Bioinformatics analysis was applied to uncover gene regulation and molecular mechanisms of CKI. Our results indicated that when associating with gefitinib in a dose-dependent fashion, CKI demonstrated the ability to inhibit the proliferation and to increase the sensitivity to gefitinib treatment in gefitinib less sensitive cell lines. This could be the results of down regulation of the PI3K/Akt/mTOR pathway and up regulation of autophagy, which were identified as the potential primary targets of CKI to increase gefitinib treatment effect.