Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Calcineurin, a Ser/Thr phosphatase linked to several signal-transduction pathways, plays a key role in the regulation of cation homeostasis, morphogenesis, cell-wall integrity, and pathogenesis in fungi. We demonstrate that calcineurin expression in S. sclerotiorum is altered in a phase-specific manner during sclerotial development. Inhibition of calcineurin by FK506, cysclosporin A, or inducible antisense calcineurin expression impaired sclerotial development at the prematuration phase and increased germination of preformed sclerotia. Induction of antisense calcineurin expression in S. sclerotiorum resulted in reduced pathogenesis on tomato and Arabidopsis. However, secretion of oxalic acid, a key virulence factor of S. sclerotiorum, was not altered. Inhibition of calcineurin conferred a reduction in cell wall β-1,3-glucan content and increased sensitivity to cell-wall-degrading enzymes and to the glucan synthese inhibitor caspofungin. Thus, calcineurin plays a major role in both sclerotial development and pathogenesis of S. sclerotiorum and, most likely, other phytopathogens.