AbstractRibosomes are ribonucleoprotein particles that are essential for protein biosynthesis in all forms of life. During ribosome biogenesis, transcription, folding, modification, and processing of rRNA are coupled to the assembly of proteins. Various assembly factors are required to synchronize all different processes that occur during ribosome biogenesis. Herein, the RNA chaperone and RNA strand annealing activity of rRNA modification enzyme ribosome small subunit methyltransferase C (RsmC), which modifies guanine to 2‐methylguanosine (m2G) at position 1207 of 16S rRNA (Escherichia coli nucleotide numbering) located at helix 34 (h34), are reported. A 25‐fold increase in the h34 RNA strand annealing rates is observed in the presence of RsmC. Single‐molecule FRET experiments confirmed the ability of protein RsmC to denature a non‐native structure formed by one of the two h34 strands and to form a native‐like duplex. This observed RNA chaperone activity of protein RsmC might play a vital role in the rapid generation of functional ribosomes.