A novel chymotrypsin inhibitor, named ClCI, was purified from coix seed (Coix lacryma-jobi L.) by aqueous two-phase extraction, chymotrypsin-Sepharose 4B affinity chromatography and centrifugal ultrafiltration. ClCI was a 7.9 kDa competitive inhibitor with pI 6.54. The inhibition constants (K_i) for bovine pancreatic chymotrypsin and bacterial subtilisin were 1.27 × 10^-10 M and 1.57 × 10^-9 M respectively. ClCI had no inhibitory activity against bovine trypsin and porcine elastase. ClCI had wide pH stability and good heat resistance. It can maintain >90% inhibition activity against chymotrypsin at 20-80 °C for 1 h. The primary structure of ClCI was highly similar (57%-92%) to those of several inhibitors belonging to the Gramineae crop potato protease inhibitor- I superfamily and showed the typical sequence motif of the protease inhibitor of the seed storage protein group. ClCI (12.5 mg) inhibited mycelial growth of the phytopathogenic fungi Mycosphaerella melonis, Helminthosporium turcicum, Alternaria solani, Phytophthora capsici, Isariopsis griseola, and Colletotrichum gloeosporioides, and caused 89% inhibition of the proteases from spore germination of plant-pathogenic fungi. The results of the present study indicate that ClCI had biotechnological potential as an alternative agent to combat the important phytopathogenic fungi.