Carnosine (β-alanyl-L-histidine) is a natural dipeptide that was first discovered in 1900 by Vladimir Gulevitch as an abundant non-protein, nitrogen-containing compound of meat. Carnosine is an archetype of a family of histidine-containing dipeptides (HCDs), and several members of this family have been identified subsequently, including anserine (β-alanyl-Nπ-methyl-L-histidine), balenine (also called ophidine, β-alanyl-Nτ-methyl-L-histidine), and homocarnosine (γ-aminobutyryl-L-histidine). Recent findings have highlighted the important roles of HCDs in muscular function and homeostasis, including their pH buffering ability, antioxidant capacity, increased Ca²⁺ sensitivity and protein glycation inhibition. The high concentration of HCDs has been observed in skeletal muscle, cardiac muscle, the brain and olfactory bulb, the stomach, and the kidneys of vertebrates. However, the biological role of carnosine and its analogues is not yet entirely known. β-alanine is a key substrate for the synthesis of carnosine, and β-alanine supplementation has been used for elevation of the carnosine content and thus has very important value in the field of medicine, pharmacy and food. Mussel is an important aquaculture shellfish in China. In order to determine the changes of small molecule metabolites and the possible metabolic mode of β-alanine supplementation in the biological body, high performance liquid chromatography-time-of-flight mass spectrometry was used to analyze the metabolite changes of Mytilus coruscus after β-alanine supplementation. Using UPLC-MS/MS technology, a total of 18 023 metabolite peaks were obtained, including 9 555 from POS model and 8 468 from NEG model, and 381 and 309 differential metabolites were therefore generated from POS model and NEG model, respectively. From these data, a set of 60 representative metabolites were screened out with Fold Change >1.3 or <0.73, and P < 0.05, after the injection of β-alanine. These significantly different metabolites were identified as carbohydrate metabolites (maltose, lactose, sucrose, glucose and fructose, etc.); amino acids and their derivatives (serine, cysteine, β-alanine, glutamine, alpha-ketoglutaric acid, etc.); lipid metabolites (photosterol, 2-2-arachidonic glycerol, etc.) and other metabolites (montane trachidine, asimidazole). Differential metabolites were then submitted to KEGG database for metabolic pathway enrichment analysis, and a total of 134 (POS mode) and 113 (NEG mode) metabolic pathways were enriched, among which, 60 representative metabolites were enriched to 25 metabolic pathways, including galactose metabolism, amino acid biosynthesis, β-alanine biosynthesis, arginine biosynthesis, glutathione metabolism, fructose and mannose metabolism, carbohydrate digestion and absorption pathways. After β-alanine injection, the free amino acids in the whole tissue of M. corucus showed that the content of 19 amino acids in the experimental group changed compared with the control group. Compared with the control group, the contents of cysteine, carnosine, and serine were significantly decreased after β-alanine injection, which was consistent with the results of the metabolomics analysis. These results revealed that β-alanine supplementation can effectively increase the energy metabolism of M. coruscus and carnosine content. This study provided a foundation for understanding the regulation of β-alanine in mussel metabolism and its mechanism, and also provided new ideas and means for improving the efficiency and nutritional value of mussel culture through β-alanine supplementation.