The molecular cloning of the exo-β-(1,3)-glucanase gene from Pichia guilliermondii K123-1 was achieved by polymerase chain reaction amplification using oligonucleotides designed according to the N-terminal amino acid sequence of purified exo-β-(1,3)-glucanase and the conserved regions in exo-β-(1,3)-glucanase from different yeast species. This gene predicts an open reading frame that has no intron and encodes a primary translation product of 408 amino acids. This preproprotein processes a mature protein of 389 amino acids by signal peptidase and a Kex2-like endoprotease. The mature protein shares 54% to 68% amino acid identity with other yeast exo-β-(1,3)-glucanases of the glycosyl hydrolase family 5. The eight invariant amino acid residues of the active site and signature pattern (IGIEALNEPL) which existed in all Family 5 members were shown in the mature protein of exo-β-(1,3)-glucanase but the fifth amino acid (LIVMGST) in the Family 5 signature pattern was changed to A. The cloned exo-β-(1,3)-glucanase gene was successfully overexpressed in Pichia pastoris X-33 and purified by Ni-NTA His-bind resin chromatography. The molecular mass of the overexpressed enzyme was determined to be approximately 44 kDa. The optimum pH and temperature for activity was 4.5 and 45oC, respectively. This enzyme showed the highest activity toward laminarin (apparent Km, 5.24 mg/mL; Vmax, 7.75 U/?g protein) among the physiological substrates and 4-methylumbelliferyl-β-D-glucoside (apparent Km, 8.67mM; Vmax, 8.99 U/?g protein) among the chromogenic substrates.