Technical Abstract: Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a Class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a Class-I and Class-II hydrophobin, respectively. To examine function, we deleted all three, separately, from the M. brunneum strain KTU-60 genome using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in pathogenicity and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and pathogenicity.