An anionic trypsin (TRY-EP) was purified from North Pacific krill (Euphausia pacifica) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography. The purified enzyme was identified as a trypsin by LC-ESI-MS/MS analysis. The relative molecular mass of TRY-EP was 33 kDa, with isoelectric point of 4.5. The histidine, tryptophan, arginine, lysine, aspartic acid and glutamic acid residues were functional groups to TRY-EP. TRY-EP was activated by Ca2+ and Mg2+ and inhibited by some heavy metal ions (Zn2+, Cu2+, Pb2+ and Hg2+), organic solvents (ethanol, glycerin, DMSO and acetone) and specific trypsin inhibitors (benzamidine, CEOM, SBTI and TLCK). TRY-EP was active over a wide pH (6.0–11.0) and temperature (10–70°C) range, with optimum of pH 9.0 and 40–50°C. TRY-EP was stable between pH 6.0 and 11.0 and below 30°C. Compared with some trypsins from the Temperate and Tropical Zone organisms, TRY-EP and other trypsins from the Frigid Zone organisms have higher affinity to substrate and 2–42-fold physiological efficiency.