RNA complexity is augmented by numerous post-transcriptional modifications, which influence RNA function by modulating its structure and interactome. One prominent modification is methylation at the ribose 2’-hydroxyl group. 2’-O-methylation has been found in all RNA classes, with rRNA and tRNA being extensively modified. The exact function of 2’-O-methylation at specific RNA sites is still not understood, with a few notable exceptions. The relevance of 2’-O-methylation for cell survival and well-being is proven by the large effort that the cell spends in maintaining a diverse and highly regulated methylation machinery. Here, we review the current knowledge on the impact of 2’-O-methylation on structure and function of different RNAs as well as on the factors determining substrate specificity in the enzymatic machinery.