Background: The adjuvanticity potential of Lactobacillus casei was first suggested in an old survey. The present study was designed to investigate the efficacy of a new immunotherapy against breast cancer made by mixing an extract of heated 4T1 mammary carcinoma cell line and a heat-killed preparation of Lactobacillus casei.Methods: Female BALB/c mice (6–8 weeks old, n=40) were challenged subcutaneously in the right flanks with 4T1 cells. When all the animals developed a palpable tumor, they were allocated to 4 equal groups and immunotherapy was initiated. The tumor-bearing mice in the experimental groups received the extract of heated 4T1 or heated Lactobacillus casei and/or a combination of both, twice at a 1-week interval. The mice in the control group received phosphate-buffered saline. One week after the last immunotherapy, one half of the mice were euthanized to determine the immune response profile. The remaining animals were kept until death occurred spontaneously.Results: The animals receiving the combined treatment significantly showed more favorable survival curves and slower rates of tumor development than the tumor-bearing mice receiving only the heated 4T1 and/or the negative control mice. The combined immunization significantly amplified the production of nitric oxide and the cytotoxicity of natural killer cells in the spleen cell culture of the tumor-bearing mice. Moreover, the combined immunotherapy significantly increased the secretion of IFN-γ and conversely diminished the secretion of IL-4 and TGF-β in the splenocyte population compared to the splenocytes from the other groups.Conclusion: The combined immunotherapy with heated 4T1 cells and heated Lactobacillus casei conferred beneficial outcomes in our mouse model of breast cancer.