Gut hormones are produced by enteroendocrine cells (EECs) found along the intestinal epithelium, and these cells play a crucial role in regulating intestinal function, nutrient absorption and food intake. A hydrolyzed casein diet has been reported to promote the secretion of gut hormones through the regulation of EEC development, but the underlying mechanism remains unclear. Therefore, this study was conducted to investigate whether the hydrolyzed casein diet can regulate EEC differentiation by employing mouse and organoid models. Mice were fed diets containing either casein (casein group) or hydrolyzed casein (hydrolyzed casein group) as the sole protein source. The hydrolyzed casein diet upregulated the expression of transcription factors, induced EEC differentiation, increased fasting serum ghrelin concentrations and promoted gastrointestinal (GI) motility in the duodenum compared to the casein diet. Interestingly, these differences could be abolished when there is addition of antibiotics to the drinking water, suggesting a significant role of gut microbiota in the hydrolyzed casein-mediated EEC function. Further investigation showed that the hydrolyzed casein diet led to reduced microbial diversity, especially the abundance of Akkermansia muciniphila (A. muciniphila) on the duodenal mucosa. In contrast, gavage with A. muciniphila impaired EEC differentiation through attenuated neurog3 transcription factor (Ngn3) expression, mediated through the promotion of Notch signaling. Moreover, pasteurized A. muciniphila showed similar effects to enter organoids in vitro. Overall, we found that a hydrolyzed casein diet reduced the abundance of A. muciniphila and promoted Ngn3 controlling EEC differentiation and this pathway is associated with increased GI motility in mice. The findings provide new insights into the role of hydrolyzed casein in gut transit and guidelines for using hydrolyzed casein in safe formula milk.