Nasulysin-1, a new zinc-metalloproteinase from the snake venom of the hognose pit viper Porthidium nasutum, was purified to homogeneity using molecular exclusion chromatography and high performance liquid chromatography on a reverse phase column. The molecular mass of the purified enzyme was 25,900 kDa and pI 4.1, as determined by 1D and 2D polyacrylamide gel electrophoresis. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the N-terminal amino acid sequence (1FSPRYIELVVVADHGMFKKYNSNLNTIR28; 1TASLANLEVWSK12; 1DLLPR6) of the purified nasulysin-1, shows close structural homology with other snake venom metalloproteinases isolated from different snake venoms. The purified nasulysin-1 showed specific apoptosis-inducing activity in Jurkat and K562 cells, a T-cell acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (AML) cell model, respectively, without affecting the viability of human lymphocyte cells. After 48 h treatment, nasulysin-1 (20 μg/mL) induced loss of the mitochondrial membrane potential (ΔΨm), activated the apoptosis-inducing factor (AIF), activated the protease caspase-3, and induced chromatin condensation and DNA fragmentation, all hallmarks of apoptosis. These results strongly suggest that nasulysin-1 selectively induces apoptosis to eliminate leukemia cells. Thus, these data warrant further investigation into the use of the metalloproteinase protein, nasulysin-1 as a potential therapeutic agent for treating leukemia.