Microcycle conidiation has been proposed as an advantageous?conidiation pattern for fungus with rapid propagation and strong resistance to stress factors. Deeply understand the microcycle conidiation mechanism will greatly enhance the potential of entomopathogenic fungi in biocontrol. Here, we isolated a microcycle conidiation associated gene, Pyk, in Metarhizium anisopliae and obtained its full length of DNA and cDNA sequence. Pyk gene in M. anisopliae encoded a homolog of pyruvate kinase with 583 amino acid residues, which showed a highly resemblance to that of other species in ascomycota (57%?77% identity). To clarify its role in microcycle conidiation, we constructed a Pyk-RNAi vector to knockdown Pyk transcript. RT-PCR demonstrated a reduced expression in mRNA level in three mutants. Furthermore, we analyzed suppression effect to Pyk in mutations. The mutants showed more varied shapes of conidia and less white hypha in colonial morphology compared to wild type.