Polynucleotide phosphorylase (PNPase) is an evolutionary conserved 3'-to-5' exoribonuclease that functions in RNA processing and turnover in prokaryotes and eukaryotes. In human, PNPase is mainly located in mitochondrial intermembrane space (IMS) where it is involved in importing a subgroup of structured RNAs into the mitochondrial matrix. Beside its IMS localization, a minor fraction of human PNPase (hPNPase) is translocate into the mitochondrial matrix, where it interacts with the helicase Suv3 to form a mitochondrial RNA exosome for RNA processing and decay in mitochondria. Mutations in hPNPase that impairs either mitochondrial RNA (mtRNA) degradation or RNA import are thus connected to mitochondrial dysfunctions and a wide spectrum of human diseases. Crystal structure of S1 truncated hPNPase reveals that this enzyme is a trimeric protein assembled into a ring-like structure with a central channel for binding of a single-stranded RNA (ssRNA) and guiding its 3' end into the active site for degradation. Here, we show that the disease-linked human PNPase mutants, Q387R and E475G, form dimers, not trimers, and have significantly lower RNA binding and degradation activities compared to wild-type trimeric PNPase. Moreover, S1 domain-truncated hPNPase binds single-stranded RNA but not the stem–loop signature motif of imported structured RNA, suggesting that the S1 domain is responsible for binding structured RNAs. We further determined the crystal structure of dimeric PNPase at a resolution of 2.8 Å, showing that the RNA-binding K homology and S1 domains are relatively inaccessible in the dimeric assembly. The overall structures of the full-length trimeric form of hPNPase was further determined by small-angle X-ray scattering (SAXS), showing that S1 domains are flexible with open to closed conformations, and that the S1 pore in the closed conformation could accommodate a stem-loop RNA. To understand how PNPase facilitates RNA import into the mitochondrial matrix, structural determination of PNPase-RNA complex by cryo-EM is underway.