The aim of this study was to explore how the mitochondrial alternative oxidase (AOX) pathway alleviates photoinhibition in chilled tomato (Solanum lycopersicum) seedlings. Chilling induced photoinhibition in tomato seedlings despite the increases in thermal energy dissipation and cyclic electron flow around PSI (CEF-PSI). Chilling inhibited the function of PSII and blocked electron transport at the PSII acceptor side, however, it did not affect the oxygen-evolving complex on the donor side of PSII. Upregulation of the AOX pathway protects against photoinhibition by improving PSII function and photosynthetic electron transport in tomato seedlings under chilling stress. The AOX pathway maintained the open state of PSII and the stability of the entire photosynthetic electron transport chain. Moreover, the protective role of the AOX pathway on PSII was more important than that on PSI. However, inhibition of the AOX pathway could be compensated by increasing CEF-PSI activity under chilling stress.