Venomous mammals are rare, and only a few species in the orders Insectivora and Monotremata produce toxic venom. Among them, the duckbill platypus (Ornithorhynchus anatinus) is one of the two venomous Australian mammals. The adult male platypus carries a spur on each hind leg, which it uses to inject competitors with poison. However, the structure and function of the poisonβs active compounds are still imcompletely characterized. We found that crude platypus venom produced potent Ca^2+^influx in human neuroblastoma IMR-32 cells. Guided by this assay, we identified 11 unique peptides, including peptide HβHisβAspβHisβProβAsnβProβArgβOH, which coincided with the N-terminal domain residues of__Ornithorhynchus__venom C-type natriuretic peptide (OvCNP). This heptapeptide induced a significant increase in [Ca^2+^]~i~in IMR-32 cells at 75 ΞΌM; had relatively specific affinities for glutamate, histamine, and GABA~A~receptors; and facilitated neurogenic twitching in guinea pig ileum specimens at 30 ΞΌM. We also established that its proteinous venom fraction strongly hydrolyzed ProβPheβArgβMCA and cleaved a human low-molecular-weight kininogen (LK), similar to porcine pancreas kallikrein. These results strongly indicated that platypus venom contains tissue kallikrein-like protease(s), and its proteolytic activity might synergistically contribute to toxicity through the specific cleavage of other venom constituents.