Metarhizium anisopliae is an important entomopathogenic fungus which is common and widely distributed in soil throughout the world. Some reports suggested that its extracellular phosphatase might play a pivotal role in invading and killing the hosts. This study explored the effect of different phosphorus sources on biomass growth and extracellular phosphatase biosynthesis of M. anisopliae by using shaking flask methods. Those evaluated phosphorus sources are including inorganic phosphorus (KH2PO4), simple organic phosphorus (phytate sodium and C6H5Na2PO4·2H2O) and protein phosphorus (casein). The results indicated that, in terms of biomass growth, production of acidic phosphatase and extracellular protein, casein yielded the best result, followed by KH2PO4 and C6H5Na2PO4·2H2O, wherein phytate sodium came at last. For purification of the extracellular protein phosphatase of M. anisopliae, the optimal phosphorus source is C6H5Na2PO4·2H2O, where there was the highest specific activity of protein phosphatase.