Type 2C Ser/Thr phosphatases (PP2Cs) are involved in various cellular processes in many eukaryotes, but little has been known about their functions in filamentous fungi. Botrytis cinerea contains four putative PP2C genes, named BcPTC1, -3, -5, and -6. Biological functions of these genes were analysed by gene deletion and complementation. While no phenotypes aberrant from the wild type were observed with mutants of BcPTC5 and BcPTC6, mutants of BcPTC1 and BcPTC3 had reduced hyphal growth, increased conidiation, and impaired sclerotium development. Additionally, BcPTC1 and BcPTC3 mutants exhibited increased sensitivity to osmotic and oxidative stresses, and to cell wall degrading enzymes. Both mutants exhibited dramatically decreased virulence on host plant tissues. All of the defects were restored by genetic complementation of the mutants with wild-type BcPTC1 and BcPTC3 respectively. Different from what is known in Saccharomyces cerevisiae, BcPtc3, but not BcPtc1, negatively regulates phosphorylation of BcSak1 (the homologue of S. cerevisiae Hog1) in B. cinerea, although both BcPTC1 and BcPTC3 were able to rescue the growth defects of a yeast PTC1 deletion mutant under various stress conditions. These results demonstrated that BcPtc1 and BcPtc3 play important roles in the regulation of multiple stress tolerance and virulence of B. cinerea.