Mitochondria of Nomuraea rileyi contain an alternative oxidase (Aox), which reduces oxygen to water by accepting electrons directly from ubiquinol. Furthermore, through a transcriptional analysis, we found that an alternative oxidase (Nraox) was up-regulated during microsclerotial formation. To study the function of NrAox, Nraox was cloned from N. rileyi CQNr01. The full-length cDNA was 1266 bp with an open reading frame of 1068 bp encoding 355 amino acids. A phylogenetic analysis revealed that the NrAox of N. rileyi was closely related to Metarhizium acridum Aox. The relative expression level of the Nraox was up-regulated during microsclerotial (MS) initiation. A salicylhydroxamic acid, a specific alternative oxidase inhibitor, application to the culture media severely decreased MS yields, changed the hyphae morphology and slowed the H2O2 removal. Nraox silencing caused mycelial deformations, reduced the MS yields by 97.3 % and increased MS size compared with those of the control. MS virulence was decreased to 26.2 % after Nraox was silenced. However, the Nraox-silenced strain was sensitive to environmental stress, and the growth rate was reduced under stress conditions. The results obtained suggested that Nraox is required for MS differentiation by regulating the intracellular H2O2 concentration and hypha growth. Additionally, Nraox had a great impact on the virulence of N. rileyi.