The late steps of both 16S and 5S ribosomal RNA maturation in the Gram-positive bacterium Bacillus subtilis have been shown to be catalysed by ribonucleases that are not present in the Gram-negative paradigm, Escherichia coli. Here we present evidence that final maturation of the 5′ and 3′ extremities of B. subtilis 23S rRNA is also performed by an enzyme that is absent from the Proteobacteria. Mini-III contains an RNase III-like catalytic domain, but curiously lacks the double-stranded RNA binding domain typical of RNase III itself, Dicer, Drosha and other well-known members of this family of enzymes. Cells lacking Mini-III accumulate precursors and alternatively matured forms of 23S rRNA. We show that Mini-III functions much more efficiently on precursor 50S ribosomal subunits than naked pre-23S rRNA in vitro, suggesting that maturation occurs primarily on assembled subunits in vivo. Lastly, we provide a model for how Mini-III recognizes and cleaves double-stranded RNA, despite lacking three of the four RNA binding motifs of RNase III.