Sea cucumber (Stichopus japonicus) autolysis during transportation and processing is a major problem and the specific proteinases responsible for autolysis have not yet been identified. In the present study, a 34 kDa serine proteinase (SP) was isolated to high purity from sea cucumber intestinal tract by a series of column chromatographies. Peptide mass fingerprinting revealed that six peptide fragments were identical to a proprotein convertase subtilisin/kexin type 9 preproprotein from sea cucumber A. japonicus. The enzyme hydrolyzed gelatin effectively at pH 6.0-9.0 and 35-40 °C, and the enzyme activity was strongly inhibited by SP inhibitors. Sea cucumber collagen was hydrolyzed significantly by purified SP at 37 °C and more gradually at 4 °C, suggesting that SP may be involved in autolysis. In addition, the SP gene that codes for 377 amino acid residues was cloned into an E. coli expression vector and expressed in vitro. A polyclonal antibody against rSP was prepared and found to react specifically against both rSP and endogenous SP, which may prove useful for future studies on the physiological functions of SP.