Plasma kallikrein (PK), a serine protease in the trypsin clan (SA), plays critical roles in many physiological and pathological pathways. Regulating the abnormal activity of PK has been successfully used in the clinical therapy of hereditary angioedema. In this study, the serine protease domain of murine plasma kallikrein (mPK) was expressed in the pichia pastoris system. The recombinant protein was a glycosylated active enzyme after purification by the cation exchange and size-exclusion chromatography, and was crystallized at the precipitant condition of 25% PEG 3350, 0.1 M Tris-HCl pH 8.5 and 0.1 M NaCl. The crystal structure of mPK was determined at 2.6 angstrom. This is the first published crystal structure of mPK, showing some distinctive features at S2' and S3' pockets when compared to its human analogue (human plasma kallikrein, hPK). In addition, mPK show unique structural features in the non-conservative 67-72 and 76-81 loops when compared to other serine proteases. These results provide insights for the design of potent and selective PK inhibitors.