Heat stress is a prevalent environmental stressor. Previous studies have shown that heat stress drives many cellular changes in Ganoderma lucidum. Interestingly, glycolysis is activated during heat stress, which could contribute to increased heat resistance. However, the molecular mechanisms underlying the enhanced heat resistance of G. lucidum following heat exposure are not yet fully understood. In this study, we explored the possibility that acetylation modification plays a significant role in responses to abiotic stress. After heat treatment, an enhanced interaction between the deacetylase GlSIRT1 and pyruvate kinase (PK) was observed, and the acetylation level of PK was decreased. Further studies revealed that GlSIRT1 increases PK activity through deacetylation, thereby increasing pyruvate content. Consistent with these findings, both PK activity and pyruvate content were reduced in GlSIRT1 knockdown strains, which exhibited greater sensitivity to heat stress compared to the wild-type (WT) strain. Collectively, our results reveal a novel molecular mechanism by which heat treatment increases pyruvate content.