Breast cancer is one of the most common cancers in women. Gut microbiome may influence tumor growth and the outcome of cancer treatment, so it may be considered as a target for tumor prevention and treatment. This study investigated the preventive and therapeutic effects of the probiotic strain, Lacticaseibacillus rhamnosus Probio-M9 (Probio-M9), against mammary cancer in mice. Thirty-six female mice were randomly divided into three groups (n = 12 per group): control group (without tumor transplantation), model group (tumor transplantation; no probiotic administration), and probiotic group (30-day oral gavage of probiotic, started seven days before tumor transplantation). Changes in tumor size was recorded, and blood, tumor tissue, and stool samples were collected at the end of the trial for analysis. Significantly smaller tumor volume was observed in the probiotic group compared with the model group (P < 0.05). Probio-M9 significantly increased the Shannon diversity index of mouse fecal microbiota and modified the gut microbiota structure (P < 0.05), characterized by significantly more Alistipes sp., Porphyromonadaceae bacterium, and Bacteroidales bacterium (P < 0.05), compared with the model group. Additionally, Probio-M9 administration elevated the serum IFN-γ, IL-9, IL-13, and IL-27 levels, while reducing the serum levels of IL-5 (P < 0.05) and several metabolites (e.g., pyridoxal, nicotinic acid, 3-hydroxybutyric acid, glutamine; P < 0.05). These physiological changes might be associated with the protective effect of Probio-M9 against mammary tumor growth. Our results support that probiotic administration could be a means of harnessing host gut microbiome and other physiological responses in combating cancer.