The enzyme polynucleotide 3′-phosphatase (EC 3.1.3.32) catalyzes the reaction

a 3′-phosphopolynucleotide + H₂O a polynucleotide + phosphate

This enzyme belongs to the family of hydrolases, specifically those acting on phosphoric monoester bonds. The systematic name is polynucleotide 3'-phosphohydrolase. Other names in common use include 2′(3′)-polynucleotidase, DNA 3′-phosphatase, deoxyribonucleate 3′-phosphatase, and 5′-polynucleotidekinase 3′-phosphatase.

As of late 2007, two structures have been solved for this class of enzymes, with PDB accession codes 1UJX and 2BRF.

• Becker A, Hurwitz J (1967). "The enzymatic cleavage of phosphate termini from polynucleotides". J. Biol. Chem. 242 (5): 936–50. doi:10.1016/S0021-9258(18)96215-0. PMID 4289819.