Abstract In this study, the anti-proliferative effect of cell-free fraction of Tibetan kefir on human gastric cancer cell line SGC7901 in vitro was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a dose-dependent antiproliferative effect, and when the concentration of Tibetan kefir cell-free fraction was more than 8 mg mL−1, a distinct anti-proliferation of SGC7901 cells was observed. Flow cytometric analysis was carried out to detect cell cycle arrest and apoptosis. SGC7901 cells treated with a cell-free fraction of Tibetan kefir were inhibited in the G1/S phase, and both early and late apoptosis could be detected. To further investigate the mechanism of action of cell-free fraction of Tibetan kefir on apoptosis of SGC7901 cells, expression of human bcl-2 and bax genes were measured by qPCR. Apoptosis induced by Tibetan kefir was effected through up-regulation of bax and down-regulation of bcl-2.