Organisms can faster and more efficiently modify and increase their thermal tolerance after a brief exposure to sublethal thermal stress. This response is called "heat-hardening" as it leads to the generation of phenotypes with increased heat tolerance. The aim of the study was to investigate the impact of heat-hardening on the metabolomic profile of M. galloprovincialis in order to identify the undergoing adjustments of biochemical pathways that might benefit mussels' thermal tolerance. Thus, mussels were exposed sequentially to two different phases (heat-hardening and acclimation phases). To gain further insights into the possible mechanisms underlying the metabolic response of the heat-hardened M. galloprovincialis, metabolomics analysis was complemented by the estimation of mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK) and alternative oxidase (AOX) implicated in the metabolic pathways of gluconeogenesis, glycolysis and redox homeostasis, respectively. Heat-hardened mussels showed evidence of higher activity of tricarboxylic acid (TCA) cycle and diversification of upregulated metabolic pathways, possibly as a mechanism to increase ATP production and extend survival under heat stress. Moreover, formate and taurine accumulation provide an antioxidant and cytoprotective role in mussels during hypoxia and thermal stress. Overall, the metabolic responses in non- and heat-hardened mussels underline the upper thermal limits of M. galloprovincialis, set at 26°C, and are in accordance with the OCLTT concept. The ability of heat-hardened mussels for a rapid gain and slow loss of heat tolerance may be an advantageous strategy for coping with the intermittent, and often extreme temperatures.