Objective:To explore the effect on proliferation and apoptosis after treatment with afatinib and Mithramycin A(MIT)in hepatocellular carcinoma cell line HepG2,and detect the aberrant expression of related factors. Methods: HepG2 cells were treated by afatinib,MIT,and combination of two. MTT and flow cytometry(FCM)were used to observe cell viability and apoptosis. EGFR,Sp1,Sp3 and genes,which were related to proliferation and apoptosis like Cyclin-D1,Cyclin-E2,Bcl-2,Caspase3,Caspase9 and p53,were analyzed by qRT-PCR. Results:With the administration time increasing,the inhibition rate of HepG2 cells apparently raised,while the group treated by afatinib and MIT exhibited dramatic ascending (P < 0.05). In addition,FCM also confirmed that Combination of afatinib and MIT arreasted HepG2 cells in G0/G1 phase,and the highest apoptosis rate appeared in combined group (P < 0.05). The level of Cyclin-D1,Cyclin-E2 and Bcl-2 in HepG2 cells incubated with both afatinib and MIT were obviously down-regulated, while Caspase3 up-regulated. Besides,afatinib enhanced expression of Caspase9 and p53,while MIT down-regulated the level of EGFR (P < 0.05). Conclusion:Afatinib combined with MIT significantly suppressed proliferation and induced apoptosis in HepG2 cells by inhibiting of Cyclin-D1,Cyclin-E2 and Bcl-2 coupled with increase of Caspase3,Caspase9 and p53. Moreover,our resultes probably provide a novel EGFR-centered strategy for future combined targeting therapy in HCC.