Protein disulfide isomerase (PDI) is a multifunctional polypeptide presents in the endoplasmic reticulum of the cell. Silkworm (Bombyx mori) pupae were used as hosts to produce recombinant PDI (rPDI). The concentration-dependent chaperone activity of rPDI was evidenced by the inhibition of the aggregation of rhodanese. Approximately 297 μg rPDI was purified from a single silkworm pupa. Results of rPDI treated with endoglycosidase H and N-glycanase, PNGase F, indicate that non-N-glycosylated rPDI (occupying 90%) and N-glycosylated rPDI are expressed in the silkworm expression system. The difference in glycosylation between silkworm pupae and yeast is discussed.