Stomata respond to darkness by closing to prevent excessive water loss during the night. Although the reorganisation of actin filaments during stomatal closure is documented, the underlying mechanisms responsible for dark-induced cytoskeletal arrangement remain largely unknown. We used genetic, physiological and cell biological approaches to show that reorganisation of the actin cytoskeleton is required for dark-induced stomatal closure. The opal5 mutant does not close in response to darkness but exhibits wild-type (WT) behaviour when exposed to abscisic acid (ABA) or CaCl2. The mutation was mapped to At5g18410, encoding the PIR/SRA1/KLK subunit of the Arabidopsis SCAR/WAVE complex. Stomata of an independent allele of the PIR gene (Atpir-1) showed reduced sensitivity to darkness and F1 progenies of the cross between opal5 and Atpir-1 displayed distorted leaf trichomes, suggesting that the two mutants are allelic. Darkness induced changes in the extent of actin filament bundling in WT. These were abolished in opal5. Disruption of filamentous actin using latrunculin B or cytochalasin D restored wild-type stomatal sensitivity to darkness in opal5. Our findings suggest that the stomatal response to darkness is mediated by reorganisation of guard cell actin filaments, a process that is finely tuned by the conserved SCAR/WAVE– Arp2/3 actin regulatory module.