Oligodendrocyte differentiation and subsequent myelination rely on strict regulatory mechanisms and molecules. Here we aimed at understanding the role of the Rho GTPase RhoA during oligodendrocyte differentiation and myelination in vivo. Using a conditional cell-specific ablation of Rhoa (Rhoa fl/fl mice crossed with Cnp-Cre mice) in oligodendrocytes, we show that RhoA controls the timing and progression differentiation and myelination. To identify putative mechanisms underlying the Rhoa cKO phenotype, we performed quantitative label-free LC-MS/MS proteomics on optic nerves from Rhoa cKO (Rhoa fl/fl Cre+) and control mice (Rhoa fl/fl Cre-) at 30 days post-birth (P30). Manual annotation of the differentially abundant proteins showed a large modulation of several proteins clusters associated with actin cytoskeleton, signal transduction and mitochondria.