<p>Environmental pH is a key factor affecting the nutritional quality of crayfish; its underlying mechanism remains unclear. This study preliminarily revealed the potential regulatory pathways underlying the effect of pH on the nutrient metabolism of crayfish through multi-omics analysis. The results showed that the contents of crude protein (18.03% vs. 16.82%), TAA (13.25% vs. 12.67%), and PUFA (55.38% vs. 45.02%) in crayfish under a high pH environment were significantly higher than those in the low pH group. Transcriptome analysis identified 1622 differentially expressed genes, among which the key gene <i>glna</i> was significantly upregulated. Metabolomics further revealed 181 differential metabolites, such as 2-phospho-<span>d</span>-glycerate and threonine, which were significantly enriched and jointly promoted the synthesis of flavor substances and amino acids. In contrast, multiple lipid-related metabolites (such as glycerone phosphate) and fat synthesis genes, such as <i>fasn</i> and <i>acsl</i>, were significantly downregulated in the low pH group, leading to reduced fatty acid content. Intestinal microbiota analysis indicated that the ratio of Firmicutes/Bacteroidota increased, and the abundance of Proteobacteria increased in the high pH group. At the genus level, the abundance of <i>Bacilloplasma</i> increased significantly. Some unclassified microbial groups in <i>Rhodobacteraceae</i> also showed enrichment. PICRUSt2 functional prediction revealed that these microbial taxa were significantly enriched in amino acid metabolism and other nutrient-related pathways, and their structural changes collectively promoted the accumulation of nutrients in the host. This study quantitatively explored the potential regulatory pathways through which the high pH environment may improve the flavor and nutritional value of crayfish at the molecular level.</p> Graphical Abstract <p></p>