Abstract Serine proteinase inhibitor Kazal-type 2 (SPINK2), a human seminal plasma proteinase inhibitor, is defined by a Kazal domain, which contains six conserved cysteine residues and forms a disulfide bridge-stabilized conformation. It has been recognized that SPINK2 is a trypsin/acrosin inhibitor involving in fertilization, however, only a few studies focused on SPINK2 during past decades. Recently, the SPINK2 gene was reported to show the greatest differential expression levels in primary cutaneous follicle center cell lymphomas (PCFCCLs). To elucidate the molecular functions of SPINK2 in non-germ cells, quantitative real-time PCR was employed by our collaborator to confirm the expression profile of SPINK2 in several leukemia cells and the invasion assays were also performed to verify the SPINK2 gene was a metastasis-enhancer in lung cancer cells. We here determined the binding site for trypsin by site-directed mutagenesis approach and the three-dimensional solution structure by nuclear magnetic resonance (NMR) spectroscopy. By using an effective strategy, recombinant SPINK proteins were successfully obtained from Escherichia coli and further employed in structural and functional analyses. By using trypsin activity assay, the results showed that SPINK2 has an ability to inhibit trypsin and the trypsin-binding region, P2-P2 site (Pro23-Arg24-His25-Phe26), was defined. Furthermore, eight point mutations within this region (P23A, P23S, P23T, R24A, H25A, H25I, H25E and F26A) were produced to determine the influences of trypsin-inhibitory activities on each site. Enzymatic kinetic studies were also carried out to calculate the inhibition constants Ki for trypsin. NMR spectroscopic results revealed that SPINK2 comprises an α-helix and one triple-stranded anti-parallel β-sheet stabilized by three disulfide bonds, which is similar to pancreatic secretory trypsin inhibitors (PSTI or SPINK1). The putative trypsin-SPINK2 complex structure was predicted by computational docking analysis. Prediction of the binding site between trypsin and SPINK2 is consistent with the results of experimental mutagenesis analysis. These studies not only provide critical information about the structural data and fundamentally biophysical features on SPINK2, but also suggest its possible effect on tumor progression and metastasis.