Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and $45^{circ}C$. The enzyme was stable at pH 4.5-6.0 and the thermal stability was up to $50^{circ}C$. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. $Cu^{2+}$, $Ni^{2+}$, and $Zn^{2+}$ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.