2′-Fucosyllactose (2′-FL), a prebiotic in human milk, has been shown to enhance the growth of Akkermansia muciniphila (A. muciniphila). Our objective is to explore the impacts of 2′-FL on the growth of A. muciniphila and the production of short-chain fatty acids (SCFAs), and its role in improving gut microbiota. In this study, our results demonstrated that 2′-FL could be used by A. muciniphila for growth, and it was metabolized into SCFAs, especially butyrate and propionate. Furthermore, the supplementation with 2′-FL led to a reduction in the Firmicutes/Bacteroidetes (F/B) ratio as well as a notable enrichment in the abundance of A. muciniphila and Muribaculaceae in mice feces. Additionally, the supplementation of 2′-FL also significantly elevated the content of SCFAs in vivo. Importantly, our findings suggested that 2′-FL might function as a prebiotic, promoting gut health by stimulating the enrichment of A. muciniphila, increasing the production of SCFAs and improving the microbiota in the gut, thereby enhancing intestinal barrier integrity, reducing intestinal inflammation and regulating immune response. Our research contributes to providing a strategy for addressing neonatal gut microbiota dysbiosis or enteritis.