The form of cancer that is prevalent among women is breast cancer and is responsible for a significant number of cancer-related deaths. Coriolus versicolor (CV) extract has demonstrated notable anti-cancer properties. Additionally, microgravity can influence various aspects of cancer cell behavior, including growth, migration, proliferation, survival, and apoptosis. This research aimed to assess the efficacy of polysaccharide peptide from CV in combination with doxorubicin under simulated microgravity (S-mu g) conditions and without S-mu g on 4T1 cancer cells. A number of 80 female inbred BALB/c mice were divided into 8 experiment groups, including Control, polysaccharide peptide from CV (T, 1 g/kg BW), doxorubicin injection (Dox, 0.5 mg/kg BW), Dox + T, S-mu g (Mic), Mic + D, Mic + T, and Mic + D + T. Subsequently, tumor biomarkers and cell death were expressed using Western blot, TUNEL assay, immunohistochemistry, and H&E staining techniques. Exposure to S-mu g reduced tumor growth compared to the control group, but its effect was less than that of the groups affected by polysaccharide peptide from CV and Dox. The Dox and CV combination could significantly (p < 0.001) inhibit 4T1 breast tumors' growth. The Dox + T group exhibited a decrease in the tumors' volume compared to control (p < 0.001). Meanwhile, it could effectively suppress tumor growth and Ki-67, VEGF, MMP-2, and MMP-9 expression. Also, Dox + T could increase the mean survival time of the tumor-bearing mice. Dox + T demonstrated a meaningful increase in cleavage of caspase-3, caspase-8, and caspase-9, confirming the occurrence of apoptosis (p < 0.001). Taking together, Dox + T can be a safe anti-cancer supplement that can cause direct apoptosis to the cancer cells. [GRAPHICS] .