Human polynucleotide phosphorylase (hPNPase) is an RNA-processing enzyme induced in response to type I interferons and during terminal differentiation and cellular senescence. hPNPase was thought to contribute to cellular senescence through its RNA-degrading activity in the cytosol; however, recent studies show that hPNPase localizes to the mitochondrial intermembrane space (IMS) and has a crucial role in maintaining mitochondrial homeostasis. Initial studies have also linked hPNPase to tumorigenesis and the cellular response to viral infection. Its surprising localization in the IMS, which is thought to be devoid of mRNA transcripts, raises questions about where and how hPNPase elicits its numerous suggested functions. Here, we discuss recent advances in understanding the various roles of hPNPase both within and potentially outside of the mitochondria.