Previously we showed that Akt-suppressing agents, combined with amrubicin, synergistically inhibited the growth of small cell lung cancer cells. The combined effects of chemotherapeutic agents and Akt-suppressing agents, including epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, were evaluated in A549 lung adenocarcinoma cells harboring K-ras mutation and wild-type EGFR. Only amrubicin and not other chemotherapeutics (cisplatin, pemetrexed and paclitaxel) synergistically inhibited cell growth when combined with an Akt inhibitor, LY294002. The combination of amrubicin and LY294002 enhanced Annexin V binding to cells. A non-specific tyrosine kinase inhibitor, genistein, suppressed Akt and showed synergistic interaction in combination with amrubicin. Two EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, suppressed Akt activity at clinically achievable concentrations and demonstrated synergism when combined with amrubicin. The suppression of K-ras expression by siRNA interfered with this synergism and inhibited both EGFR and Akt activity in A549 cells. In Ma10 cells, which harbor wild-type EGFR and K-ras, EGFR-TKIs neither suppressed Akt activity nor exhibited such synergism when combined with amrubicin. We concluded that the synergism by the combination of EGFR-TKI and amrubicin is attributable, at least partially, to K-ras mutation in A549 cells. The combination of EGFR-TKI and amrubicin may be a promising treatment for lung cancer with wild-type EGFR and K-ras mutation.