The anti-cancer effects of heat-killed Enterococcus faecium KU22001 (KU22001), KU22002, and KU22005 isolated from human infant feces were investigated. The anti-proliferative activity of these strains against various cancer cell lines was evaluated using the MTT assay. To determine the production of exopolysaccharides (EPS) with potential anti-cancer effect, ethanol precipitation and phenol-sulfuric acid method was used with the cell free supernatant of strains grown at 25℃ or 37℃. The EPS yield of E. faecium strains was higher at 25℃ than at 37℃. Among these E. faecium strains, KU22001 grown at 25℃ was associated with the highest bax/bcl-2 ratio, effective apoptosis rate, cell cycle arrest in the G0/G1 phase, and condensation of the nucleus in the cervical cancer HeLa cell line. In conclusion, these results suggest that KU22001 can be beneficial owing to the anti-cancer effects and production of functional materials, such as EPS.