Melanoma differentiation-associated-7 (mda-7)/interleukin-24 (IL-24), a unique cytokine-tumor suppressor, exerts tumor-selective killing activity in numerous types of cancer cell. Although eukaryotically and prokaryotically expressed recombinant human (rh)IL-24 proteins have been previously shown to produce potent antitumor effects, to the best of our knowledge, no side-by-side study has been conducted that compares the two proteins directly. In the present study, rhIL-24 protein was expressed in BL21 Escherichia coli transformed with the pET-21a(+)-hIL-24 plasmid by isopropyl-beta-D-1-thiogalactopyra noside induction. Following a denaturing and renaturing process, the soluble rhIL-24 was purified using a Q-Sepharose column. rhIL-24 protein was also expressed in Chinese hamster ovary mammalian cells stably transfected with the pcDNA3-hIL-24 plasmid. The in vitro antitumor efficacies of the two treatments were compared using the MDA-MB-231 human breast cancer cell line. Furthermore, the therapeutic efficacies of the bacteria-derived rhIL-24 protein and the liposome-coated pcDNA3-hIL-24 naked plasmid were evaluated in athymic nude mice with subcutaneously xenografted MDA-MB-231 cell tumors. The prokaryotically expressed/purified rhIL-24 protein and the eukaryotically expressed rhIL-24 in the cell supernate were revealed to be capable of efficiently suppressing MDA-MB-231 tumor growth in vitro. Similarly, the administration of bacteria-derived rhIL-24 protein and pcDNA3-hIL-24 naked plasmid also provided therapeutic benefits in the treatment of in vivo MDA-MB-231 xenografted tumors. The retarded in vitro and in vivo breast cancer growth elicited by rhIL-24 was closely associated with the upregulation of the ratio of anti-apoptotic B cell lymphoma 2 (Bcl-2) to pro-apoptotic Bcl-2-associated X protein (Bax), as well as the activation of caspase-3 followed by marked induction of apoptosis, and the notable inhibition of tumor angiogenesis. Thus, the results of the present study indicate that prokaryotically expressed rhIL-24 protein may be an alternate and promising antitumor agent in human breast cancer or other types of cancer.