5′-Nucleotidase (5′-NT) is widely represented in animal tissues (CD73) as well as in almost all snake venoms. In the present study, a 5′-NT isoform has been isolated from Vipera lebetina venom. The homodimeric isoform consists of monomers with molecular masses of 60 kDa. The enzyme is thermolabile and has pH optimum at 7.5. The 5′-NT activity is inhibited by metal ions Fe3+, Cu2+ and Zn2+, enhanced by Mn2+ while Mg2+ and Ca2+ have no remarkable effect. In addition to 120-kDa protein there are higher molecular forms of 5′-NT present in the V. lebetina venom. The cloning and sequencing of the 5′-NT coding cDNA resulted in 5′-truncated construct. MALDI-TOF and Orbitrap mass-spectrometry of the tryptic peptides confirmed the translated N-terminally truncated protein sequence concordance to the 5′-NT isolated from the venom. The isolated protein strongly inhibited ADP- or collagen-induced platelet aggregation.