Heat hardening in Mytilus galloprovincialis is a survival strategy for enhancing thermal tolerance in response to extreme low tides during consecutive low-tide periods in spring. Transcriptome sequencing was used to analyze the gill tissues of mussels in the heat-hardening (H) and control (N) groups to uncover the mechanisms of thermal tolerance enhancement under heat hardening. The results showed that, compared to the control group, the heat-hardening group had 651 differentially expressed genes, including 159 significantly upregulated and 492 significantly downregulated genes. Gene ontology (GO) analysis revealed significant enrichment of differentially expressed genes in biological processes such as metabolism, substance transport, and signal transduction. Gene set enrichment analysis (GSEA) of significantly enriched items in the GO analysis revealed the upregulation of biological processes related to macromolecule metabolism, nucleic acid metabolism, and metabolism of compounds containing nuclear bases, which are associated with macromolecule metabolism, substance metabolism, and energy metabolism. Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that differentially expressed genes were significantly enriched in signaling pathways related to metabolic regulation, such as Rap1, apoptosis-multiple species, and MAPK. These findings indicate that thermal hardening in Mytilus galloprovincialis may enhance tissue functional capacity by activating various macromolecular metabolic processes while also strengthening the ability of gill tissues to actively remove damaged cells, thus enabling early physiological regulation and adaptive responses to subsequent high-temperature stress, leading to better functional performance. This study explored the biological processes and metabolic pathways associated with thermal hardening in Mytilus galloprovincialis, providing a foundation for investigating the molecular mechanisms underlying heat-hardening in this species.