Abstract: Objective To explore the molecular mechanism of sorafenib resistance in hepatoma cells and identify for new targets to reverse drug resistance. Methods THP-1 cells were induced into M2 tumor-associated macrophages (M2-TAMs) in vitro and identified by immunofluorescence. SMMC-7721 cells were co- cultured with M2-TAMs with or without sorafenib treatment. CCK-8 assay was used to observe the inhibitory effect of sorafenib on the cell proliferation. Annexin V/PI double staining and protein immunoblotting were used to assess the effect of sorafenib on the proliferation, apoptosis and the expressions of apoptosis-related proteins and autophagy-related protein in SMMC-7721 cells co-cultured with M2-TAMs in the presence or absence of the autophagy inhibitor chloroquine (CQ). Results The IC50 of sorafenib at 48 h was 2.25 μmol/L in SMMC-7721 cells cultured alone, and increased to 4.72 μmol/L in the cells co-cultured with M2-TAMs. Compared with the cells cultured alone, the co-cultured SMMC-7721 cells showed significantly reduced apoptosis rate in response to sorafenib (P<0.01) and significantly increased expression of Bcl-2 and Bcl-2/Bax ratio (P<0.05) with also increased LC3-II/LC3- I ratio (P<0.001) and lowered expression of p62 (P<0.05), suggesting a significantly enhanced level of autophagy. CQ treatment significantly inhibited the proliferation of the co-cultured SMMC-7721 cells (P<0.05), increased the cell apoptosis (P<0.05) and reduced the Bcl-2/Bax ratio (P<0.01). Conclusion M2-TAMs can attenuate the inhibitory effect of sorafenib on the proliferation of hepatoma cells by increasing the level of autophagy, suggesting a new strategy for reversing sorafenib resistance induced by the tumor microenvironment by inhibiting autophagy.