Objective To obtain a chimera composed of annexin B1 (AnxB1) and melittin (MLT) and to investigate its inhibitory effect on phosphatidylserine liposome activity and SMMC7721 and HepG2 cell proliferation. Methods A fusion gene AnxB1-MLT was constructed by overlap extension gene splicing and then was inserted into plasmid pGEX-5T. The recombinant plasmid was transformed into E. coli strain K802 and induced by IPTG at low temperature. The expression condition was optimized and GST affinity chromatography column was used for purification. Calcium-dependent phospholipid binding assay was used to determine whether the chimera kept the activity of AnxB1. CCK8 analysis was employed to investigate the effect of AnxB1-MLT on SMMC7721 and HepG2 cell proliferation. Results After being inserted into the expression plasmid, AnxB1-MLT protein expressed in K802 cells, with a high level recombinant protein induced by 0.2 mmol/L IPTG at 22-24℃ for 4 h. The protein AnxB1-MLT was purified by using GST affinity chromatography column (a band at 63 000) and the purification of the final purified protein was >95%. AnxB1-MLT was able to bind to phosphatidylserine liposome in a calcium-dependent manner. CCK8 analysis indicated that AnxB1-MLT inhibited SMMC7721 and HepG2 cell proliferation at a dose-dependent manner. Conclusion We have successfully constructed a chimera AnxB1-MLT, which retains the calcium-dependent phospholipid binding activity of AnxB1, and can inhibit the proliferation of hepatic cancer cell lines SMMC7721 and HepG2.