Background and aim: Nowadays, the probiotic bacteria such as lactobacilli are known as prevention factor for various disease especially cancer. The aim of this study was to investigate the cytotoxic effect of Lactobacillus casei PTCC 1608 cell extract as probiotic bacteria on colon cancer cell line (HT29) and analysis of Bax and Bcl2 apoptosis gene expression. Methods: In this experimental study, the cell extract of heat killed L. casei was prepared in 0.01, 0.1, 1, 10, 100 and 1000 µg/ml concentration and subsequently, the cytotoxicity of various cell extracts on HT29 and HEC293 cell lines were evaluated in 24 hours using MTT assay. Moreover, the Bax and Bcl2 apoptosis gene expression level in HT29 cell line was analyzed using Real Time PCR. The apoptotic effects of cell extract was determined using Flow-cytometry technique. Finally, the collected data were statistically analyzed using one-way anal­ysis of variance with the SPSS/18 software. Results: The results of MTT test show that cell extracts of L. casei is able to reduce the survival rate of HT29 cell line to 0.95±0.44, 73.45±0.21, 51.49±0.87, 39.5±0.45 and 19.7±0.55. In addition to, the Real Time PCR results indicated the expression level of Bax and Bcl2 was increased and decreased respectively, in HT29 cell line (2.76 ± 0.54 (P<0.05), 0.21 ± 0.43 (P< 0.05)) in 24 h. Moreover, the flow cytometry results indicated the 35.62 % apoptosis in HT29 cell line treated with IC50 value. Conclusion: The results show that the cell extract of L. casei PTCC 1608 could induced the apoptosis in HT29 cell line and it had low toxicity on HEC293 cell line. Therefore, it seems that L. casei has potential uses as probiotic for pharmaceutical applications including prevention and treatment of colon cancer.