Previously, we showed that ascorbate (Asc), by donating electrons to photosystem II (PSII), supports a sustained electron transport activity in heat-treated leaves. In this study, by using wild-type, Asc-overproducing and -deficient Arabidopsis thaliana mutants (miox4 and vtc2–3, respectively), we investigated the physiological role of Asc as PSII electron donor in heat-stressed leaves (40 °C, 15 min). Chl a fluorescence transients show that PSII reaction centers became gradually inactivated upon illumination following heat stress, a process which was initiated by a dramatic deceleration of the electron transfer from TyrZ to P680+ and was followed by the complete loss of charge separation activity. These processes strongly depended on the Asc content of leaves: At 300 μmol photons m−2 s−1 they occurred with halftimes of 1.2 and 10 min, 2.8 and 23 min, and 4.1 and 51 min in vtc2–3, the wild type and miox4, respectively. Photoinactivation was slowed down by diphenylcarbazide, an artificial PSII electron donor. Western blot analysis shows that in addition to D1, CP43 and PsbO are also degraded. Our data provide strong evidence for the alleviation of donor-side induced photoinhibition by ascorbate by acting as alternative electron donor to PSII.