Background and Objective: Breast cancer, assumed as a difficult -to –treat disorder across the world, is the most common malignancy among women. Over decades, significant advances in breast cancer immunotherapy and tumor immune biology have been brought about. In vitro studies of the effect of various cytokines have been conducted in breast tumor. Among Interleukins, IL-27 -a novel cytokine- is associated with specific properties. Moreover, IL-27 contributes to the Th1 induction and also acts as a pro- inflammatory cytokine. The aim of this study was to evaluate the IL-27 anti-proliferative effects on 4T1 cell line in vitro. Materials and Methods: In this study, 4T1 cells were cultured in RPMI1640. mIL-27 gene was cloned, cells were transfected and recombinant protein was produced. Then, anti-proliferative effect of IL-27 on 4T1 breast cancer cell line was evaluated. Results: Our results indicated that IL-27 could suppress 4T1 cell proliferation significantly (p<0/01). Cell to cell interactions and also morphology of the cells were remarkably changed in comparison to control cells. Conclusion: Our results showed that, IL-27 under in vitro conditions, could potentially suppress tumor without any essential cells and biologic factors of tumor matrix. Therefore, rmIL-27 may be a probable candidate protein as an antitumor agent, applicable to breast cancer immunotherapy.