Lactobacilli are indigenous microbes of the stomach of rodents, with much lower numbers being present in mice fed a purified diet than in those fed a non-purified diet. We postulated that gastric emptying (GE) is responsible for the different colonisation levels of lactobacilli and tested this hypothesis in the present study. BALB/cCr Slc mice were fed either a non-purified diet or a purified diet for 2 weeks. The number of gastric tissue-associated lactobacilli was lower in mice fed the purified diet than in those fed the non-purified diet. GE, estimated by measuring the food recovered from the stomach, was higher in mice fed the purified diet than in those fed the non-purified diet and correlated negatively with the number of lactobacilli. Mice fed the non-purified diet exhibited lower GE rates even when lactobacilli were eliminated by ampicillin administration through the drinking-water, suggesting that GE is the cause but not the consequence of different Lactobacillus colonisation levels. The plasma concentrations of acylated ghrelin, a gastric hormone that promotes GE, were higher in mice fed the purified diet than in those fed the non-purified diet. There was a negative correlation between GE and the number of lactobacilli in mice fed the non-purified diet, the purified diet, and the purified diet supplemented with sugarbeet fibre (200 g/kg diet) or carboxymethyl cellulose (40 g/kg diet). We propose that a higher GE rate contributes, at least in part, to lower gastric colonisation levels of lactobacilli in mice fed a purified diet.