Stomatal opening is stimulated by red and blue light. Blue light activates plasma membrane (PM) H+-ATPase by phosphorylating its penultimate residue, threonine, via a blue light photoreceptor phototropin-mediated signaling pathway in guard cells. Blue light-activated PM H+-ATPase promotes the accumulation of osmolytes and, thus, the osmotic influx of water into guard cells, driving stomatal opening. Red light-induced stomatal opening is thought to be dependent on photosynthesis in both guard cell chloroplasts and mesophyll cells; however, how red light induces stomatal opening and whether PM H+-ATPase is involved in this process have remained unclear. In this study, we established an immunohistochemical technique to detect the phosphorylation level of PM H+-ATPase in guard cells using whole leaves of Arabidopsis (Arabidopsis thaliana) and unexpectedly found that red light induces PM H+-ATPase phosphorylation in whole leaves. Red light-induced PM H+-ATPase phosphorylation in whole leaves was correlated with stomatal opening under red light and was inhibited by the plant hormone abscisic acid. In aha1–9, a knockout mutant of one of the major isoforms of PM H+-ATPase in guard cells, red light-dependent stomatal opening was delayed in whole leaves. Furthermore, the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibited red light-induced PM H+-ATPase phosphorylation as well as red light-induced stomatal opening in whole leaves. Our results indicate that red light-induced PM H+-ATPase phosphorylation in guard cells promotes stomatal opening in whole leaves, providing insight into the photosynthetic regulation of stomatal opening.