We examined the impact of Lactobacillus casei subsp. casei 327 (Lc327) on colonic production of serotonin (5-hydroxytryptamine, 5-HT), a critical regulator of gastrointestinal (GI) function. Heat-killed form of Lc327 was orally administered to BALB/c mice for 14 consecutive days. Significantly higher amounts of 5-HT were detected in colonic tissues from Lc327 group mice compared to control mice. Immunohistochemistry assays revealed that significantly higher frequency of the cells containing 5-HT markedly in colon from Lc327 group mice. In accordance with these findings, elevation in relative mRNA levels for tryptophan hydroxylase 1, the rate-limiting enzyme for mucosal 5-HT, was observed in colonic epithelium from Lc327 group mice. Additionally, oral administration of heat-killed Lc327 shortened the transit time of colonic bead expulsion in mice. These results suggest that the heat-killed Lc327 has a potential to promote 5-HT synthesis in colonic mucosa, which could contribute to improving GI motility.