Aim The aim of this study was to investigate whether two Lactobacillus reuteri strains (rat‐derived R2LC and human‐derived ATCC PTA 4659 (4659)) could protect mice against colitis, as well as delineate the mechanisms behind this protection. Methods Mice were given L. reuteri R2LC or 4659 by gavage once daily for 14 days, and colitis was induced by addition of 3% DSS (dextran sulphate sodium) to drinking water for the last 7 days of this period. The severity of disease was assessed through clinical observations, histological evaluation and ELISA measurements of myeloperoxidase (MPO) and pro‐inflammatory cytokines from colonic samples. Mucus thickness was measured in vivo with micropipettes, and tight junction protein expression was assessed using immunohistochemistry. Results Colitis severity was significantly reduced by L. reuteri R2LC or 4659 when evaluated both clinically and histologically. The inflammation markers MPO, IL‐1β, IL‐6 and mKC (mouse keratinocyte chemoattractant) were increased by DSS and significantly reduced by the L. reuteri strains. The firmly adherent mucus thickness was reduced by DSS, but significantly increased by L. reuteri in both control and DSS‐treated mice. Expression of the tight junction proteins occludin and ZO‐1 was significantly increased in the bottom of the colonic crypts by L. reuteri R2LC. Conclusion These results demonstrate that each of the two different L. reuteri strains, one human‐derived and one‐rat‐derived, protects against colitis in mice. Mechanisms behind this protection could at least partly be explained by the increased mucus thickness as well as a tightened epithelium in the stem cell area of the crypts.