Dietary betaine supplementation has been reported to alleviate the adverse effects of high-carbohydrate diets on Megalobrama amblycephala, while the regulatory mechanism remains largely unknown. In the present study, a 79-day feeding trial was conducted with 450 juvenile Megalobrama amblycephala (average weight 6.75 ± 0.10 g), which were fed with five high-carbohydrate diets (43%) supplementing betaine at 0% (CD group), 0.2% (0.2Bet group), 0.4% (0.4Bet group), 0.8% (0.8Bet group), and 1.6% (1.6Bet group), respectively. Results showed M. amblycephala in 0.8Bet group exhibited the best growth performance, indicated by the largest weight gain ratio (142.88%) and least feed conversion ratio (1.63). Moreover, liver health was promoted in 0.8Bet group, with decreased number of non-nucleated cells and less lipid accumulation, which was accompanied by the lowest hepatosomatic index (1.38%). In order to further illustrate the regulatory mechanism, metabolites assay indicated that dietary betaine supplementation significantly increased plasma contents of methionine, serine, hypotaurine, and taurine, but did not affect plasma contents of cystathionine, cystine, or cysteic acid. Accordingly, the mRNA expressions of cysteine sulfinate decarboxylase in cysteine sulfinic acid pathway and cysteamine dioxygenase (ADO) in sulfinic acid (CS) pathway, which were both involved in taurine synthesis, were also upregulated in the liver. Meanwhile, the microbial communities in M. amblycephala intestine were more stable and uniform with betaine supplementation. Therefore, dietary betaine supplementation may exert its protective roles in improving feed efficiency and liver health of M. amblycephala via promoting de novo taurine synthesis and stabilizing intestinal microbial communities.