In our previous work, we characterized the in vitro biofunctionality and evaluated the biosafety of exopolysaccharide (EPS)-producing Weissella confusa VP30 (VP30), newly isolated from healthy children's feces.Since the puri ed EPS (pEPS) from fermented milk with VP30 (VP30-EPS) showed signi cant water holding capacity, it was theorized that its consumption would relieve constipation. In this work, the in vivo functionalities of VP30-EPS and pEPS were evaluated for their effect on constipation using an experimental constipated rat model. Rats were randomly divided into four groups: (i) negative control (PBS administered normal group), (ii) loperamide treated positive control (constipation group), (iii) constipation with loperamide plus VP30-EPS (1 g/kg) and (iv) constipation with loperamide plus pEPS (0.6 g/kg) groups. Loperamide treatment induced animal constipation and signi cantly reduced the frequency of defecation, intestinal transit ratio, and water content of feces. However, all four fecal parameters were improved in both the loperamide plus VP30-EPS and pEPS administered groups as compared to the loperamide treated positive control group. No signi cant changes in dietary intake or serum hepatocellular necrosis maker levels were observed in any experimental group. These results suggest that the addition of VP30-EPS or pEPS potentially improves the functional laxative effects of commercial products. No other published research relating in vivo functional effects of EPS from Weissella spp. on constipation could be identi ed. This study suggests the possibility that VP30-EPS and pEPS can be applied to fermented and/or functional foods to relieve constipation.