Calcium signaling plays important roles in stress tolerance and virulence in fungi. Mid1, an accessory protein of Cch1 calcium channel, has been discussed in baker's yeast and some filamentous fungi. However, functions of the Mid1 gene in entomopathogenic fungi are not clear. In this study, the Mid1 gene was functionally characterized by deleting it in the entomopathogenic fungus Metarhizium acridum. The growth of the MaMid1 mutant was similar as the wild type on normal growth medium, but inhibited by exogenous Ca2+, Fe2+, Mg2+, Mn2+, Li+, and calcium chelator ethylene glycol tetraacetic acid (EGTA). Cation transportation-related genes were upregulated and intracellular calcium concentration was decreased in MaMid1. Deletion of the MaMid1 gene impaired the tolerance to cell wall-disrupting agents but had no impact on heat or ultraviolet irradiation tolerance compared with the wild type. Bioassays showed that MaMid1 had decreased virulence, with defects in the ability to penetrate the host cuticle. Compared with the wild type, appressorium formation on locust wings and fungal growth in the insect hemocoel were significantly decreased in the MaMid1 mutant in a bioassay through topical inoculation. The phenotypes of MaMid1 were fully restored in a complementation strain. Taken together, our study demonstrates that the MaMid1 affects intracellular ion homeostasis and contributes to virulence by affecting the initial penetration process in M. acridum.