Intestinal microbiota plays a crucial role in the nutritional metabolism and immune functions of animals. In order to investigate the impact of two cultivation modes, namely grass-based shrimp farming and paddy field shrimp farming, on the intestinal microbiota and muscle flavor of Macrobrachium rosenbergii, this experiment focused on specimens raised in different cultivation areas with the same breeding cycle. The specimens were categorized into a grass-based cultivation group (Grass Group) and a paddy field cultivation group (Paddy Group). The study conducted analyses on the structure of intestinal microbiota, muscle amino acids, and trace elements in the muscle content. Firstly, at the phylum level classification of shrimp intestinal microbiota, Firmicutes, Proteobacteria, and Tenericutes were identified as dominant phyla under both cultivation modes (with a relative abundance > 5%). Additionally, Cyanobacteria and Bacteroidetes were identified as specific dominant phyla in the Grass Group. At the genus level classification, Lactococcus, Enterobacter, and Candidatus_Hepatoplasma were identified as dominant genera in both groups. Secondly, in terms of microbiota diversity analysis, the number of operational taxonomic units (OTUs) in the Paddy Group was significantly higher than that in the Grass Group, indicating a higher species diversity of intestinal microbiota in Macrobrachium rosenbergii under paddy field cultivation. The functional prediction of the intestinal microbiota revealed that the functions in both cultivation modes were mainly concentrated in metabolic regulation pathways. Finally, in the analysis of nutritional components and the evaluation of muscle flavor, it was proved that there was no significant difference in the total contents of umami amino acids and sweet amino acids between them. However, the Paddy Group exhibited significantly higher levels of serine, glycine, and arginine in the muscle compared to the Grass Group. The iron content in the muscle of Macrobrachium rosenbergii under paddy field cultivation was significantly higher, while copper and zinc content were significantly lower compared to the Grass Group. There were no significant differences in the content of other mineral elements. In conclusion, Macrobrachium rosenbergii under paddy field cultivation exhibited superior muscle flavor and a more diverse intestinal microbiota. The results of this study can provide technical references and guidance for different health cultivation modes of Macrobrachium rosenbergii.