The high-osmolarity glycerol signal pathway plays an important role in the response of fungi to various environmental stresses. In this study, we characterized a mitogen-activated protein kinase kinase kinase gene BcOS4 in Botrytis cinerea, which is homologous to Saccharomyces cerevisiae SSK2/SSK22. The BcOS4 deletion mutant was significantly impaired in vegetative growth and conidial formation. The mutant exhibited increased sensitivity to the osmotic, oxidative stresses and to the fungicides iprodione and fludioxonil. Western blot analysis showed that BcSak1, a putative downstream component of BcOs4, was not phosphorylated in the mutant. In addition, the BcOS4 mutant was unable to infect leaves of rapeseed and cucumber, and grape fruits, although it can cause disease on apple fruits. All the defects were restored by genetic complementation of the BcOS4 deletion mutant with the wild-type BcOS4 gene. The data of this study indicate that BcOS4 is involved in vegetative differentiation, virulence, adaption to hyperosmotic and oxidative stresses, and to fungicides in B. cinerea.