To express bovine chymosin in yeast, we amplified the prochymosin gene from the plasmid pMD18T-Prochy by PCR, and then cloned the gene into the expression vector pPICZaA, resulting in pPICZaA-Prochy. Pichia pastoris GS115 was used as host cells. Integration of the prochymosin cDNA into the Pichia pastoris genome was confirmed by PCR and sequencing analysis. Chymosin was expressed in Pichia pastoris successfully, and a strong band at about 37 kD was shown by SDS-PAGE. Activity tests showed that the chymosin activity of the culture supernatant was 12.2 SU/mL. This is the first report of successful expression of chymosin in Pichia pastoris. The recombinant Pichia pastoris strain obtained in this study could be further used to produce recombinant chymosin for cheese making.