Verticillium saksenae A-1 was newly isolated and identified, as a nematophagous fungus, from a cucumber cultivating field, Gurye, Korea. V. saksenae A-1reduced the reproduction of pine wood nematode Bursaphelenchus mucronatus and was parasitic to the eggs and juvenile bodies of nematodes. Over 50% of nematodes were immobilized by V. saksenae A-1 culture filtrate within 48 h exposure. It was confirmed that the culture filtrate of V. saksenae A-1contained chitinase, chitosanse, and protease. A 30 kDa protease was purified from its culture filtrate by Sephadex G-150 gel filtration and DEAE-Sephadex A-50 anion exchange chromatography. The anti-nematicidal activity of A-1 protease against pine wood nematode Bursaphelenchus mucronatus was confirmed and the mortality was over 60% in 48 hrs according to the protease treatment under the described condition. When treated with the protease and 0.01% Fluorescent Brightener 28, the inner part of nematode body showed more brilliant than the one treated with the heated protease, mainly due to digestion and damage of the cuticle layer of nematode body by the protease. These results indicate that the A-1 protease is responsible for the nematicidal activity of V. saksenae A-1, a newly isolated nematophagous fungus, against B. mucronatus.