The eukaryotic calcineurin (CN) pathway comprising catalytic A (CnA) and regulatory B subunits (CnB) is crucial for many biological processes but functionally unexplored in entomopathogenic fungi. Here, we characterise three CN subunits (CnA1, CnA2 and CnB) and a downstream CN-responsive zinc finger transcription factor (Crz1) in Beauveria bassiana. CN-mediated phosphatase activity decreased by 16-38 % in all deletion mutants compared with wild type. Growth and conidiation were most defective in Delta cnB, which showed a large proportion of abnormally branched germlings but were less defective in Delta cnA1 and Delta cnA2. Conidiation defects also occurred in Delta crz1, uniquely accompanied with slower germination. Compared with wild type, the four deletion mutants became, to varying degrees, more sensitive to Ca2+, Mn2+, Zn2+, Mg2+, two oxidants, three cell wall stressors, carbendazim, heat shock and ultraviolet (UV)-B irradiation. They were also less virulent to Spodoptera litura larvae. Only Delta cnB and Delta crz1 were less tolerant to high osmolarity. The altered phenotypes of the deletion mutants were associated with lower intracellular mannitol and trehalose levels, reduced overall activity of superoxide dismutases and catalases, altered cell wall composition and down-regulation of numerous phenotype-influencing genes. Additionally, the transcription of six cascaded genes in two stress-responsive mitogen-activated protein kinase (MAPK) pathways and the phosphorylation of hallmarking Hog1 and Slt2 were largely down-regulated in all the deletion mutants under osmotic and cell wall stresses, respectively. All the changes were restored by gene complementation. Taken together, three calcineurin subunits and Crz1 play vital, but variable, roles in B. bassiana responses to environmental stresses during development and host signals during infection.