High temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S‐nitrosoglutathione reductase (GSNOR) activity and expression were down‐regulated with the simultaneous accumulation of total S‐nitrosothiols (SNOs) including S‐nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO2‐Tyr) studied by high‐performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine‐nitrated proteins. Among the induced proteins, ferredoxin–NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3‐morpholinosydnonimine (SIN‐1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.