This study sought to uncover whether icotinib can enhance bufalin-induced apoptosis of human colon cancer cells and the roles of PI3K/Akt signaling pathway in the apoptosis. The cell proliferation of human colon cancer cell lines RKO, HT29, CACO-2 and SW480 treated by bufalin was detected by MTT assay. Then, the apoptosis rate of RKO and SW480 cells treated by bufalin alone and combination of bufalin and icotinib was detected by flow cytometry. Afterwards, the levels of Cbl-b, p-AKT, p-ERK, PARP, Bax and Bcl-2 in RKO and SW480 cells were determined by Western blotting. Additionally, the effect of K-Ras silencing on the synergy of bufalin and icotinib was assessed. Bufalin decreased RKO, HT29, CACO-2 and SW480 (EX12 mutation) cell viability in a dose-dependent manner and induced apoptosis of RKO and SW480 cells in vitro. In the cells treated by combination of bufalin and icotinib, the levels of Cbl-b, cleaved PARP and Bax were increased, while the levels of p-Akt and Bcl-2 were reduced, comparing with that in the bufalin treated cells. Furthermore, K-Ras silencing (including EX12 mutated K-Ras and wild-type K-Ras) did not significantly affect the apoptosis rate of cells treated by the combination of bufalin and icotinib. Icotinib synergizes with bufalin to induce the apoptosis of colon cancer cells through PI3K/Akt signal pathway and regulating the apoptosis related proteins (Bcl-2, Cbl-b, cleaved PARP and Bax). K-Ras may not participate in the combination of bufalin and icotinib induced apoptosis in human colon cancer cells.