The microbial community residing in the intestine (microbiota) is an important environmental factor regulating nutrient metabolism and energy storage. An improved understanding of the dynamic relationships between host, microbiota and diet could lead to novel therapies for metabolic diseases. The objective of this study is to define the role of the gut microbiota and diet on zebrafish nutrient digestion. We compared in vivo digestive physiology of gnotobiotic zebrafish larvae exposed to different microbial and dietary stimuli, by feeding fluorescent reporters of phospholipase A2 (PED6) and protease activity (EnzChek). Colonization of germ‐free zebrafish at 3 days post‐fertilization (dpf) with a normal microbiota resulted in increased PED6 and EnzChek digestion within 24 hours independent of diet. In vitro assays revealed that gut microbes can contribute directly to PED6 and EnzChek processing. We find that microbial stimulation of PED6 and EnzChek digestion in vivo is not associated with altered feeding behavior, but is dependent on exogenous nutrients by 6dpf. Our results indicate that microbial and nutritional signals interact to stimulate digestive enzyme activity in the zebrafish gut. Future experiments will determine if microbial and nutritional status influences production or release of pancreatic enzymes, and test the role of specific nutrients and bacterial genes. NIH DK073695 and DK081426