Melanoma is a metastatic type of skin cancer with an increasing prevalence rate worldwide. Lactobacillus rhamnosus (L. rhamnosus) is a probiotic with valuable biological and pharmaceutical effects. To introduce a novel approach for melanoma treatment, we aimed to investigate the effect of L. rhamnosus on the growth and survival of mouse melanoma cells both in vitro and in vivo. B16-F10 and L929 cells were treated with serial dilutions of the heat-killed cell-free supernatant (CFS) of L. rhamnosus (1:10, 1:5, and 1:2.5) for four consecutive days. Cell proliferation was evaluated using alamarBlue and clonogenic assay. Flow cytometry was carried out using propidium iodide and annexin V–fluorescein isothiocyanate. For in vivo studies, B16-F10 cells were subcutaneously injected into C57BL/6 mice, and tumor volume, relative tumor volume, and tumor growth inhibition were calculated upon the injection of heat-killed CFS of L. rhamnosus. Findings indicated significant (p < 0.001) reduction in the proliferation of B16-F10 cells upon treatment with the heat-killed CFS of L. rhamnosus, while no toxic effect was observed in normal fibroblasts. Results of clonogenic assay indicated significant (p < 0.0001) decrease in the survival fraction of B16-F10 cells, and flow cytometry analysis revealed no noticeable apoptosis-inducing effects. Results of in vivo analysis indicated significant (p < 0.01) decrease in tumor growth upon the injection of heat-killed CFS of L. rhamnosus into mouse melanoma models. In conclusion, the present study provides the first report that heat-killed CFS of L. rhamnosus inhibits B16-F10 melanoma cell growth in vitro and reduces tumor growth in vivo. To better evaluate observed therapeutic effects of this probiotic, more research on human melanoma cell lines is recommended.

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