Background: The anti-cancer activity of some lactic acid bacterial strains is well documented in several kinds of literatures. Lactobacillus strains have received considerable attention as a beneficial microbiota. The aim of this study is to evaluate the effects of anti-tumor activities of L. acidophilus ATCC4356 culture supernatants on the MCF-7 human breast cancer cells. Materials and Methods: The anti-cancer effects of 24h and 48h culture supernatants at various concentrations (1.25, 2.5, 5, 10 and 20 µg/ml) were determined by various in vitro and in vivo assays including MTT, tumor volume measurement as well as99mTc-MIBI biodistribution in MCF-7 tumor bearing nude mice and histopathology test. For evaluation of the related mechanism of action, quantitative PCR was conducted. Results: The 48h culture supernatants at 10 and 20 µg/ml exhibited significant in vitro inhibition of MCF-7 cell proliferation. However, this inhibition was not observed for HUVEC human endothelial normal cells. Q-PCR indicated that treatment by the supernatant led to a significant downregulation of VEGFR (~ 0.009 fold) and Bcl-2 (~ 0.5 fold) and upregulation of p53 (~ 1.3 fold). In vivo study using MCF-7 xenograft mouse models demonstrated a reduction in tumor weight and volume by both 24h and 48h supernatants (2 mg/kg) after 15 days. According to the99mTc-MIBI biodistribution result, treatment of MCF-7 bearing nude mice with both 24h and 48h supernatant (2mg/kg) led to a significant decrease in tumor uptake compared with the control group. Conclusion: These results suggest that the culture supernatants of L. acidophilus ATCC4356 at suitable concentrations can be considered as a good alternative nutraceutical with promising therapeutic indexes for breast cancer.