Colon cancer is one of the most common cancer around the world. Exopolysaccharides (EPSs) produced by lactobacilli as potential prebiotics have been found to have an anti-tumor effect. In this study, lyophilized EPSs of four Lactobacillus spp. for their impact on apoptosis in colon cancer cells (HT-29) was evaluated using flow cytometry. The relationship between capability of a lactobacilli-EPS to induce apoptosis and their monosaccharide composition, molecular weight (MW), and linkage type was investigated by HPLC, SEC, and NMR, respectively. Changes in apoptotic-markers were examined by qPCR and Western Blotting. EPSs were capable of inhibiting proliferation in a time-dependent manner and induced apoptosis via increasing the expression of Bax, Caspase 3 and 9 while decreasing Bcl-2 and Survivin. All EPSs contained mannose, glucose, and N-acetylglucosamine with different relative proportions. Some contained arabinose or fructose. MW ranged from 10(2)-10(4)Da with two or three fractions. EPS of L. delbrueckii ssp. bulgaricus B3 having the highest amount of mannose and the lowest amount of glucose, showed the highest apoptosis induction. In conclusion, lactobacilli-EPSs inhibit cell proliferation in HT-29 via apoptosis. Results suggest that a relationship exists between the ability of EPS to induce apoptosis and its mannose and glucose composition.