Objective To prepare recombinant lidamycin (rLDM) and study its antitumor activity. Methods The ldp gene was cloned in the experssion vector pET30a(+) and induced in E.coli. The recombinant lidamycin apoprotein rLDP was purified by Ni2+ affinity chromatography. rLDM was prepared by reloading the AE of lidamycin into the rLDP. The cytotoxicity of rLDM and LDM was examined by MTT assay. The analysis of cell cycle was examined by flow cytometry. Results The experssion vector pET30-sldp was constructed, and rLDP was successfully secreted into the culture medium and periplasmic space of E.coli. HPLC showed that rLDP-AE had absorption at 340 nm, meaning rLDM had been reconstituted successfully. The results of MTT assay showed that the IC50 value of rLDM for SKOV3 cells was close to that of LDM. FACS analysis of cell cycle showed that rLDM and LDM induced similar cell cycle arrest in SKOV3 cells. Conclusion Recombinant lidamycin apoprotein rLDP was successfully constructed and expressed in E.coli, rLDM was obtained by molecular reconstitution. As compared with the natural LDM, rLDM shows corresponding activity to cancer cells.