Malate synthase (Mls), a key enzyme in the glyoxylate cycle, is required for virulence in microbial pathogens. In this study, we identified the AoMls gene from the nematode-trapping fungus Arthobotrys oligospora. The gene contains 4 introns and encodes a polypeptide of 540 amino acids. To characterize the function of AoMls in A. oligospora, we disrupted it by homologous recombination, and the Delta AoMls mutants were confirmed by PCR and Southern blot analyses. The growth rate and colony morphology of the Delta AoMls mutants showed no obvious difference from the wild-type strains on potato dextrose agar (PDA) plate. However, the disruption of gene AoMls led to a significant reduction in conidiation, failure to utilize fatty acids and sodium acetate for growth, and its conidia were unable to germinate on minimal medium supplemented with sodium oleate. In addition, the trap formation was retarded in the Delta AoMls mutants, which only produced immature traps containing one or two rings. Moreover, the nematicidal activity of the Delta AoMls mutants was significantly decreased. Our results suggest that the gene AoMls plays an important role in conidiation, trap formation and pathogenicity of A. oligospora.