In fungi, the Ca2+/calcineurin signaling pathway is critical in mediating growth, morphology, stress responses, and pathogenicity. Crz1 is a calcineurin-responsive zinc finger transcription factor. Here, MaCrz1 was identified and functionally characterized in the entomopathogenic fungus Metarhizium acridum by characterization of a targeted gene knockout strain. Conidia of the Delta Macrz1 mutant were aberrant in cell surface features and lacked the characteristic hydrophobic rodlet layer, and the mutant displayed increased sensitivity to oxidative stress, cell wall perturbing agents, heat stress, and ultraviolet irradiation as compared to the wild-type and complemented strains. Insect bioassay using locusts revealed decreased virulence for the Delta MaCrz1 mutant, with defects in the ability of the mutant to penetrate the host cuticle. The Delta MaCrz1 mutant also showed greatly reduced chitin and beta-1,3-glucan level in the cell wall. Transcriptomic profiling revealed genes involved in cell wall synthesis, conidiation, stress tolerance, and calcium transport that were downregulated in the Delta MaCrz1 mutant. Our results demonstrate that MaCrz1 plays important roles in stress susceptibility and pathogenicity, and provides clues as to the genes and pathways targeted by the transcription factor.