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Phototropins are light-activated receptor kinases that mediate a wide range of blue light responses responsible for the optimization of photosynthesis. Despite the physiological importance of phototropins, it is still unclear how they transduce light signals into physiological responses. Here, we succeeded in reproducing a primary step of phototropin signaling in vitro using a physiological substrate of phototropin, the BLUS1 (BLUE LIGHT SIGNALING1) kinase of guard cells. When PHOT1 and BLUS1 were expressed in Escherichia coli and the resulting recombinant proteins were incubated with ATP, white and blue light induced phosphorylation of BLUS1 but red light and darkness did not. Site-directed mutagenesis of PHOT1 and BLUS1 revealed that the phosphorylation was catalyzed by phot1 kinase. Similar to stomatal blue light responses, the BLUS1 phosphorylation depended on the fluence rate of blue light and was inhibited by protein kinase inhibitors, K-252a and staurosporine. In contrast to the result in vivo, BLUS1 was not dephosphorylated in vitro, suggesting the involvement of a protein phosphatase in the response in vivo. phot1 with a C-terminal kinase domain but devoid of the N-terminal domain, constitutively phosphorylated BLUS1 without blue light, indicating that the N-terminal domain has an autoinhibitory action and prevents substrate phosphorylation. The results provide the first reconstitution of a primary step of phototropin signaling and a clue for understanding the molecular nature of this process.