Cocoon, a shelter for larva development to silk moth, contains the fibrous protein fibroin, which is coated by the globular protein sericin. Emergence of the silk moth requires the action of cocoonase, a protease secreted by the pupa. The full-length prococoonase cDNA, with 780 bp open reading frame encoding 260 amino acids, was cloned by reverse transcription from total RNA of the head of 6-day-old Thai-silk Bombyx mori pupa. Only the gene fragment lacking the propeptide encoding sequence was successfully expressed in Pichia pastoris, yielding an extracellularly active cocoonase. The recombinant cocoonase was purified to homogeneity by 80% ammonium-sulfate fractionation and CM-Sepharose chromatography, and its internal peptide sequences were analyzed by nano liquid chromatography-mass spectrometry/mass spectrometry. This monomeric protein has native molecular weight of 26 kDa by gel exclusion analysis and 25 kDa subunit size by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme hydrolyses sericin but does not hydrolyse fibroin, as shown by radial diffusion on thin-layer enzyme assay (RD-TEA). Scanning electron microscopy showed that purified recombinant cocoonase could remove sericin from natural silk completely in 24 h, without damaging fibroin, using only 1 immobilized sericin unit (ISU) of enzyme as determined by RD-TEA. Natural cocoonase isolated from B. mori pupa could also digest sericin effectively, but required more enzymes (2 ISU) and longer time (48 h). In comparison, a commercial enzyme, alcalase, with the same activity not only showed less complete digestion of sericin but also caused damage of fibroin. These results suggest that recombinant B. mori cocoonase is potentially useful for silk degumming.