The essential photoprotective role of proton gradient regulation 5 (PGR5)-dependent cyclic electron flow (CEF) has been reported in Arabidopsis, rice, and algae. However, its functional assessment has not been performed in tomato yet. In this study, we focused on elucidate the function of SlPGR5 and SlPGR5-like photosynthetic phenotype 1 (PGRL1) in tomato. We performed RNA interference and found that SlPGR5/SlPGRL1-suppressed transformants exhibited extremely low CO2 assimilation capacity, their photosystem I (PSI) and PSII were severely photoinhibited and chloroplasts were obviously damaged. The SlPGR5/SlPGRL1-suppressed plants almost completely inhibited CEF and Y(ND), and PSII photoinhibition may be directly related to the inability to produce sufficient proton motive force to induce NPQ. The transgenic plants overexpressing SlPGR5 and SlPGRL1 driven by 35S promoter capable alleviate photoinhibition of plants under low night temperature. The transcriptomic and proteomic analyses suggested that the nuclear gene transcription and turnover of chloroplast proteins, including the plastoglobule-related proteins, were closely related to SlPGR5/SlPGRL1 pathway dependent CEF. The bridge relationship between CEF and chloroplast quality maintenance was a novel report to our knowledge. In conclusion, these results revealed the regulatory mechanism of the SlPGR5/SlPGRL1 pathway in photoprotection and maintenance of chloroplast function in tomato, which is crucial for reduce yield loss, especially under adverse environmental conditions.