Significance The presence of RNA chemical modifications has long been known, but their precise molecular consequences remain unknown. 2′- O -methylation is an abundant modification that exists in RNA in all domains of life. Ribosomal RNA (rRNA) represents a functionally important RNA that is heavily modified by 2′- O -methylations. Although abundant at functionally important regions of the rRNA, the contribution of 2′- O -methylations to ribosome activities is unknown. By establishing a method to disturb rRNA 2′- O -methylation patterns, we show that rRNA 2′- O -methylations affect the function and fidelity of the ribosome and change the balance between different ribosome conformational states. Our work links 2′- O -methylation to ribosome dynamics and defines a set of critical rRNA 2′- O -methylations required for ribosome biogenesis and others that are dispensable.