Polynucleotide phosphorylase (PNPase) is an exoribonuclease that catalyzes the processive phosphorolytic degradation of RNA from the 3′‐end. The enzyme catalyzes also the reverse reaction of polymerization of nucleoside diphosphates that has been implicated in the generation of heteropolymeric tails at the RNA 3′‐end. The enzyme is widely conserved and plays a major role in RNA decay in both Gram‐negative and Gram‐positive bacteria. Moreover, it participates in maturation and quality control of stable RNA. PNPase autoregulates its own expression at post‐transcriptional level through a complex mechanism that involves the endoribonuclease RNase III and translation control. The activity of PNPase is modulated in an intricate and still unclear manner by interactions with small molecules and recruitment in different multiprotein complexes. Not surprisingly, given the wide spectrum of PNPase substrates, PNPase‐defective mutations in different bacterial species have pleiotropic effects and perturb the execution of genetic programs involving drastic changes in global gene expression such as biofilm formation, growth at suboptimal temperatures, and virulence. WIREs RNA 2016, 7:241–258. doi: 10.1002/wrna.1328This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Turnover and Surveillance > Regulation of RNA Stability