Enterotoxigenic Escherichia coli (ETEC) is a leading cause of infectious diarrhea in children and postweaning piglets. ETEC infection results in induced pro-inflammatory responses in intestinal epithelial cells and dysbiosis of intestinal microbiota. Here, a Lactobacillus reuteri strain, HCM2, isolated from a healthy piglet showed a high survival rate in the harsh gastrointestinal tract environment and inhibited the growth of ETEC and its adherence to intestinal epithelial cells. Pre-supplementation with L. reuteri HCM2 for 14 days reduced the ETEC load in the jejunum of ETEC-infected mice and prevented the disruption of intestinal morphology by ETEC. The colonic microbiota of mice with or without HCM2 pre-supplementation were analyzed, and this analysis revealed that HCM2 could prevent dysbiosis caused by ETEC infection by stabilizing the relative abundance of dominant bacteria. These results indicate that L. reuteri HCM2 has the potential to attenuate the effect of ETEC on the colonic microbiota in infected mice.