Our previous study showed that the overexpression of carboxypeptidase Y (CPY) of Saccharomyces cerevisiae in Escherichia coli resulted in the formation of insoluble inclusion bodies. To produce soluble CPY, we designed a novel Pichia pastoris expression system, in which the following were inserted into expression vectors: three different signal sequences derived from the mating factor a1 of S. cerevisiae, an inulinase of Kluyveromyces marxianus, and the endogenous signal sequence of CPY. The expression vector pHIL-D2-SSinul-proCPY was the most effective in the production of proCPY among the vectors examined. The purified active CPY was obtained from proCPY by treating with proteinase K, followed by QExcellose ion-exchange column chromatography.