Probiotic bacteria are frequently used to treat intestinal diseases or to improve health; however, little is known about the evolutionary changes of these bacteria during probiotic manufacture and the bacterial ability to colonize the intestine. It has been observed that when bacteria adapt to a new environment, they lose some traits required to thrive in the original niche. In this study, a strain of Lactobacillus reuteri was isolated from mouse duodenum and subjected to 150 serial passes in milk to simulate the industrial propagation of probiotic bacteria. The strains adapted to milk outperformed their ancestor when grown in milk; we also showed evidence of reduced intestinal colonization of milk-adapted strains. Whole-genome sequencing showed that bacterial adaptation to milk selects mutants with altered metabolic functions.