The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP synthesis in plants. The PMF mainly arises from the oxidation of water in photosystem II and from electron transfer within the cytochrome b 6 f complex. There are two electron transfer pathways related to PMF formation: linear electron flow and cyclic electron flow. Proton gradient regulation 5 (PGR5) is a major component of the cyclic electron flow pathway, and the Arabidopsis pgr5 mutant shows a substantial reduction in the PMF. How the PGR5-dependent cyclic electron flow contributes to ATP synthesis has not, however, been fully delineated. In this study, we monitored in vivo ATP levels in Arabidopsis chloroplasts in real time using a genetically encoded bioluminescence-based ATP indicator, Nano-lantern(ATP1). The increase in ATP in the chloroplast stroma of pgr5 leaves upon illumination with actinic light was significantly slower than in wild type, and the decrease in ATP levels when this illumination stopped was significantly faster in pgr5 leaves than in wild type. These results indicated that PGR5-dependent cyclic electron flow around photosystem I helps to sustain the rate of ATP synthesis, which is important for growth under fluctuating light conditions.