Proteomic analyses of human nucleoli provided molecular bases for an understanding of the multiple functions fulfilled by these nuclear domains. However, the biological roles of about 100 of the identified proteins are unpredictable. The present article describes the functional characterization of one of these proteins, ISG20L2. We demonstrate that ISG20L2 is a 3' to 5' exoribonuclease involved in ribosome biogenesis at the level of 5.8S rRNA maturation, more specifically in the processing of the 12S pre-rRNA. The use of truncated forms of ISG20L2 demonstrated that its N-terminal half promotes the nucleolar localization and suggested that its C-terminal half bears the exoribonuclease activity. Identification of the binding partners of ISG20L2 confirms its involvement in the biogenesis of the large ribosomal subunit. These results strongly support the notion that, in human, as it was demonstrated in yeast, 5.8S rRNA maturation requires several proteins in addition to the exosome complex. Furthermore, this observation sustains greatly the idea that the extremely conserved need for correctly processed rRNAs in vertebrates and yeast are achieved by close but different mechanisms.