Probiotic bacteria have been studied for their potential impact on the metabolism of dietary components in the small intestine lumen including lactose digestion, metabolism of lipids such as cholesterol, and oxalate metabolism. In the large intestine, they contribute to the metabolism of otherwise indigestible dietary carbohydrates (e.g., prebiotics) and have a favorable effect on colonic protein and ammonia metabolism, although their effect on the digestive fate of phytochemicals and xenobiotics is still uncertain. Probiotics also influence metabolism in the host tissues, in particular the gastrointestinal mucosa and the liver. Underlying mechanisms include supply of additional enzymatic activities in the gut lumen and alterations of the composition or metabolic pattern of the gut resident microbiota. For future studies, selection of probiotic strains should include assessment of their metabolic activities, and the outcome of the intervention studies should also take into account the composition of the probiotic matrix and the background diet of the target population. New technologies such as metabolomics hold great promise for assessment of probiotics functionality.