Simple SummaryThis study used 16S rRNA sequencing and metabolomic methods to investigate the key gut microbiota and metabolites associated with the growth performance ofMacrobrachium rosenbergiifamilies. We found that some critical intestinal bacteria, includingLactobacillusandBlautia, and the metabolites related to metabolism of amino acids and fatty acids may play critical roles in the growth of prawns. This study contributes to figuring out the landscape of the gut microflora and intestinal metabolites associated with prawn growth performance and provides a basis for future studies on the probiotic feed of prawns.AbstractTo investigate the key gut microbiota and metabolites associated with the growth performance ofMacrobrachium rosenbergiifamilies, 16S rRNA sequencing and LC–MS metabolomic methods were used. In this study, 90M. rosenbergiifamilies were bred to evaluate growth performance. After 92 days of culture, high (H), medium (M), and low (L) experimental groups representing three levels of growth performance, respectively, were collected according to the weight gain and specific growth rate of families. The composition of gut microbiota showed that the relative abundance of Firmicutes, Lachnospiraceae,Lactobacillus,andBlautiawere much higher in Group H than those in M and L groups. Meanwhile, compared to the M and L groups, Group H had significantly higher levels of spermidine, adenosine, and creatinine, and lower levels of L-citrulline. Correlation analysis showed that the abundances ofLactobacillusandBlautiawere positively correlated with the levels of alpha-ketoglutaric acid and L-arginine. The abundance ofBlautiawas also positively correlated with the levels of adenosine, taurine, and spermidine. Notably, lots of metabolites related to the metabolism and biosynthesis of arginine, taurine, hypotaurine, and fatty acid were upregulated in Group H. This study contributes to figuring out the landscape of the gut microbiota and metabolites associated with prawn growth performance and provides a basis for selective breeding.