The stomatal pores of higher plants enable gaseous exchange into and out of leaves for photosynthesis and evaporation. Stomatal opening is induced by both blue and red lights. It is shown that blue light-induced stomatal opening is mediated by the blue light receptor phototropins (PHOT1 and PHOT2) and cryptochromes (CRY1 and CRY2). However, whether phytochrome B (phyB) is involved in red light regulation of stomatal opening remains largely unclear. Here, we report a positive role for Arabidopsis (Arabidopsis thaliana) phyB in the regulation of red light-induced stomatal opening. The phyB mutant stomata displayed a reduced red light response, whereas stomata of the phyB-overexpressing plants displayed a hypersensitive response to red light. In addition, stomata of the cry1 cry2 phyB, phot1 phot2 phyB, and cry1 phyA phyB triple mutant plants showed more reduced light response than those of the single or double mutant plants under white light, implying that phyB acts in concert with phyA, CRY, and PHOT in light regulation of stomatal opening. Stomata of phyB cop1 mutant opened less wide than those of the cop1 mutant, and stomata of the pif3 pif4 mutant opened wider than those of the wild-type, indicating that COP1, together with the PIFs (phytochrome interacting factors), may act downstream of PHYB in regulating stomatal opening. Furthermore, quantitative RT-PCR analysis showed that the expression of MYB60 was reduced in the cry1 cry2 and phyA phyB mutants under blue and red lights, respectively, but induced in the CRY1- and phyB-overexpressing plants. These results demonstrate that phyB and CRY might regulate stomatal opening, at least in part, by regulating MYB60 expression.