The secreted phospholipases A 2 (sPLA 2 s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A 2 (hsPLA 2 -IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA 2 -IID in Escherichia coli, the DNA-coding sequence for hsPLA 2 -IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA 2 -IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A 2 . The refolded recombinant hsPLA 2 -IID demonstrated Ca 2+ -dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA 2 -IID which will advance our understanding of the structure–function relationship and biological effects of the protein.