Phototropins are autophosphorylating protein kinases of plant-specific blue light receptors. They regulate various blue light responses, including phototropism, chloroplast movements, hypocotyl growth inhibition, leaf flattening, and stomatal opening. However, the physiological role of autophosphorylation remains unknown. Here, we identified phosphorylation sites of Ser or Thr in the N terminus, Hinge1 region, kinase domain, and C terminus in Arabidopsis phototropin1 (phot1) by liquid chromatography–tandem mass spectrometry in vivo . We substituted these Ser or Thr residues with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses of stomatal opening, phototropism, chloroplast accumulation, and leaf flattening after the transformation of the phot1 phot2 double mutant. Among these sites, we found that autophosphorylation of Ser-851 in the activation loop of the kinase domain was required for the responses mentioned above, whereas the phosphorylation of the other Ser and Thr, except those in the activation loop, was not. Ser-849 in the loop may have an additional role in the responses. Immunological analysis revealed that Ser-851 was phosphorylated rapidly by blue light in a fluence-dependent manner and dephosphorylated gradually upon darkness. We conclude that autophosphorylation of Ser-851 is a primary step that mediates signaling between photochemical reaction and physiological events.