Soy foods are known to be effective for breast cancer prevention. The habitual consumption of soy isoflavones in combination with the probiotic Lactobacillus casei Shirota (LcS) was shown to decrease the risk of breast cancer occurrence in our previous population-based case-controlled study among Japanese women. The present study aimed to elucidate the cooperative prevention mechanism of soymilk and LcS using an animal carcinogenic model. Female Sprague–Dawley rats received a high-fat, AIN-76A diet containing soymilk, LcS, both soymilk and LcS, or none and were orally exposed to 2-amino-1-methyl-6-penylimidazo[4,5-b]pyridine at a dose of 85 mg/kg bodyweight eight times for 2 weeks. The development of palpable mammary tumors was monitored for 17 weeks. Tumor tissues were immunohistochemically examined for estrogen receptor (ER)-α, Ki-67 and CD34. Compared with the control group, the incidence and multiplicity of mammary tumors were reduced by soymilk alone and soymilk in combination with LcS, while tumor volume was decreased by LcS alone and LcS in combination with soymilk. An immunohistochemical analysis revealed that soymilk in combination with LcS more effectively reduced the numbers of ER-α-positive and Ki-67-positive cells in tumors than soymilk alone and that both soymilk and LcS inhibited tumor angiogenesis. These results demonstrated that soymilk prevents the development of mammary tumors and that LcS suppresses tumor growth, potentially enhancing the preventive efficacy of soymilk. The habitual consumption of LcS in combination with soymilk might be a beneficial dietary style for breast cancer prevention.