Heat stress impairs both pollen germination and pollen tube elongation, resulting in pollination failure caused by energy imbalance. Invertase plays a critical role in the maintenance of energy homeostasis; however, few studies investigated this during heat stress. Two rice cultivars with different heat tolerance, namely TLY83 (heat tolerant) and LLY722 (heat susceptible) were subjected to heat stress. At anthesis, heat stress significantly decreased spikelet fertility, accompanied by notable reductions in pollen germination on stigma and pollen tube elongation in ovule, especially in LLY722. Acid invertase (INV), rather than sucrose synthase contributed to sucrose metabolism, which explains the different tolerances of both cultivars. Under heat stress, larger enhancements in NAD(H), ATP, and antioxidant capacity were found in TLY83 compared with LLY722, while a sharp reduction in Poly (ADP-ribose) polymerase (PARP) activity was found in the former compared with the latter. Importantly, exogenous INV, 3-aminobenzamide (a PARP inhibitor), sucrose, glucose, and fructose significantly increased spikelet fertility under heat stress, where INV activity was enhanced and PARP activity was inhibited. Therefore, INV can balance the energy production and consumption to provide sufficient energy for pollen germination and pollen tube growth under heat stress. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.