Updated project metadata. Elevated temperatures due to global warming seriously threaten crops production. Understanding molecular mechanisms of cellular responses to heat stress will help to improve crop tolerance to high temperature and yield. In this work, we show that deacetylation of non-histone proteins mediated by the rice cytoplasmic histone deacetylase HDA714 is required for plant tolerance to heat stress. HDA714 expression and protein accumulation are induced by heat stress. HDA714 loss-of-function affects specifically plant response to heat (42°C) while its over-expression enhances plant tolerance to the stress. Interestingly, heat stress led to decreases of overall protein lysine acetylation in rice plants, which depends on HDA714 function. HDA714-mediated deacetylation of metabolic enzymes stimulates glycolysis under heat stress. In addition, HDA714 protein is found within heat-induced stress granules (SGs), many identified rice SG proteins show lysine acetylation at normal temperature (25 °C), which is augmented in hda714 mutants. Finally, HDA714 interacts with and deacetylates several SG proteins and HDA714 loss-of-function impairs SG formation. Collectively, these results indicate that HDA714-mediated cellular protein lysine deacetylation responds to heat stress, affects metabolic activities, regulates SGs formation, and confers heat tolerance in rice plants.