Polynucleotide phosphorylase (PNPase) is a diverse enzyme, involved in RNA polyadenylation, degradation, and processing in prokaryotes and organelles. However, in human mitochondria, PNPase is located in the intermembrane space (IMS), where no mitochondrial RNA (mtRNA) is known to be present. In order to determine the nature and degree of its involvement in mtRNA metabolism, we stably silenced PNPase by establishing HeLa cell lines expressing PNPase short-hairpin RNA (shRNA). Processing and polyadenylation of mt-mRNAs were significantly affected, but, to different degrees in different genes. For instance, the stable poly(A) tails at the 3′ ends of COX1 transcripts were abolished, while COX3 poly(A) tails remained unaffected and ND5 and ND3 poly(A) extensions increased in length. Despite the lack of polyadenylation at the 3′ end, COX1 mRNA and protein accumulated to normal levels, as was the case for all 13 mt-encoded proteins. Interestingly, ATP depletion also altered poly(A) tail length, demonstrating that adenylation of mtRNA can be manipulated by indirect, environmental means and not solely by direct enzymatic activity. When both PNPase and the mitochondrial poly(A)-polymerase (mtPAP) were concurrently silenced, the mature 3′ end of ND3 mRNA lacked poly(A) tails but retained oligo(A) extensions. Furthermore, in mtPAP-silenced cells, truncated adenylated COX1 molecules, considered to be degradation intermediates, were present but harbored significantly shorter tails. Together, these results suggest that an additional mitochondrial polymerase, yet to be identified, is responsible for the oligoadenylation of mtRNA and that PNPase, although located in the IMS, is involved, most likely by indirect means, in the processing and polyadenylation of mtRNA.