Various stress conditions induce the nuclear translocation of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), but its nuclear function in plant stress responses remains elusive. Here we show that GAPC interacts with a transcription factor to increase the expression of heat-inducible genes and heat tolerance in Arabidopsis. GAPC accumulates in the nucleus in a heat stress-dependent manner. Screening of Arabidopsis transcription factors identifies nuclear factor Y subunit C10 (NF-YC10) as a GAPC-binding protein. Heat tolerance of seedlings and the expression of heat-inducible genes are enhanced by overexpression of GAPC and reduced in gapc. The effects of GAPC overexpression are abolished when NF-YC10 is deficient or GAPC nuclear accumulation is suppressed. Genetic complementation fails to recover the heat responses of gapc when GAPC-NF-YC10 interaction is disrupted. GAPC overexpression also enhances the binding ability of NF-YC10 to its target promoter. The results reveal a cellular and molecular mechanism for the nuclear moonlighting of a glycolytic enzyme in plant response to environmental changes.