Opening of stomata in the plant facilitates photosynthetic CO2 fixation and transpiration. Blue-light perception by phototropins (phot1, phot2) activates the plasma membrane H(+)-ATPase, causing stomata to open. Here we describe a regulator that connects these components, a Ser/Thr protein kinase, BLUS1 (BLUE LIGHT SIGNALING1), which mediates a primary step for phototropin signalling in guard cells. blus1 mutants identified by infrared thermography result in a loss of blue light-dependent stomatal opening. BLUS1 encodes a protein kinase that is directly phosphorylated by phot1 in vitro and in vivo at Ser-348 within its C-terminus. Both phosphorylation of Ser-348 and BLUS1 kinase activity are essential for activation of the H(+)-ATPase. blus1 mutants show lower stomatal conductance and CO2 assimilation than wild-type plants under decreased ambient CO2. Together, our analyses demonstrate that BLUS1 functions as a phototropin substrate and primary regulator of stomatal control to enhance photosynthetic CO2 assimilation under natural light conditions.