5′-Nucleotidase (EC 3.1.3.5) is an enzyme which catalyzes the phosphorylytic cleavage of 5′-nucleotides. Although originally found in snake venom, the activity of 5'nucleotidase has been described for bacteria and plant cells, and is widely distributed in vertebrate tissue. In mammalian cells the enzyme is predominantly located in the plasma membrane and its primary role is in the conversion of extracellular nucleotides (e.g. 5'-AMP), which are generally impermeable, to the corresponding nucleoside (e.g. adenosine) which can readily enter most cells. Consequently, the enzyme plays a key role in the metabolism of nucleotides.

The enzyme has a wide substrate specificity for nucleotides and has been shown to hydrolyze 5'nucleotides rapidly, ribose-5-phosphate slowly, and other phosphate esters extremely slowly (if at all).

The enzyme catalyses the following reaction:

a 5′-nucleotide + H₂O ⇌ a nucleoside + phosphate

The 5′-nucleotidase-catalyzed reaction of an AMP nucleotide to adenosine nucleoside is shown below: