The molecular cloning of the exo-$eta$-(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-$eta$-(1,3)-glucanase and the conserved regions in exo-$eta$-(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-$eta$-(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-$eta$-(1,3)-glucanase but the fifth amino acid (LIVMGST) in the Family 5 signature pattern was changed to A. The cloned exo-$eta$-(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 $45^{circ}C$, respectively. This enzyme showed the highest activity toward laminarin (apparent Km, 5.24 mg/mL; Vmax, $7.75;U/{mu}g$ protein) among the physiological substrates and 4-methylumbelliferyl-$eta$-D-glucoside (apparent Km, 8.67 mM; Vmax, $8.99;U/{mu}g$ protein) among the chromogenic substrates.