Purpose: The universal organic solvent dimethyl sulfoxide (DMSO)can be used as a differentiation inducer of many cancer cells andhas been widely used as a solvent in laboratories. However, itseffects on breast cancer cells are not well understood. The aimof this study is to investigate the effect and associated mechanismsof DMSO on mouse breast cancer. Methods: We appliedDMSO to observe the effect on tumors in a mouse breast cancermodel. Tumor-associated macrophages (TAMs) were tested byflow cytometry. Ex vivo tumor microenvironment was imitated by4T1 cultured cell conditioned medium. Enzyme-linked immunosorbentassays were performed to detect interleukin (IL)-10 andIL-12 expression in medium. To investigate the cytotoxicity ofDMSO on TAMs, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assays were performed. Results: We foundthat DMSO produced tumor retardation when injected into mouseperitoneal cavities in a certain concentration range (0.5–1.0 mg/g). Furthermore, as detected by flow cytometry, TAM subtypeswere found to be transformed. We further imitated a tumor microenvironmentin vitro by using 4T1 cultured cell conditionedmedium. Similarly, by using low concentration DMSO (1.0%–2.0% v/v), TAMs were induced to polarize to the classically activatedmacrophage (M1-type) and inhibited from polarizing intothe alternatively activated macrophage (M2-type) in the conditionedmedium. IL-10 expression in tumors was reduced, whileIL-12 was increased compared with the control. Furthermore, wereported that 2.0% (v/v) DMSO could lead to cytotoxicity in peritonealmacrophages after 48 hours in MTT assays. Conclusion:Our findings suggest that DMSO could exert antitumor effects in4T1 cancer-bearing mice by reversing TAM orientation and polarizationfrom M2- to M1-type TAMs. These data may providenovel insight into studying breast cancer immunotherapy.