The role ofMetarhizium robertsiiGroup III histidine kinase (mhk1) in regulating various phenotypes of the fungal entomopathogen and the transcripts of 25 downstream genes likely associated with the phenotypes were probed by constructing Δmhk1and Δmhk1/mhk1mutants. All examined Δmhk1phenotypes except unchanged sensitivity to fungicide (dimethachlon) differed significantly from those of wild type and Δmhk1/mhk1, which were similar to each other. Significant phenotypic changes in Δmhk1included increased conidial yields on two media, increased tolerance to H2O2, decreased tolerance to menadione, increased tolerance to hyperosmolarity, increased conidial thermotolerance, decreased conidial UV-B resistance and reduced virulence toTenebrio molitorlarvae. Themhk1disruption elevated the transcripts of nine genes, including two associated with conidiation (flbCandhymA) and three encoding catalases but decreased seven other gene transcripts, including three for superoxide dismultases, under normal conditions. The high-osmolarity glycerol pathway MAPK phosphorylation level in Δmhk1culture was increased 1.0- to 1.8-fold by KCl, sucrose and menadione stresses but reduced drastically by H2O2 or heat (40°C) stress, accompanied with different transcript patterns of all examined genes under the stresses. Our results confirmed the crucial role of mhk1 in regulating the expression of the downstream genes and associated phenotypes important for the fungal biocontrol potential.