Myelination depends on timely, precise control of oligodendrocyte differentiation and myelinogenesis. Cholesterol is the most abundant component of myelin and essential for myelin membrane assembly in the central nervous system. However, the underlying mechanisms of precise control of cholesterol biosynthesis in oligodendrocytes remain elusive. In the present study, we found that Qki depletion in neural stem cells or oligodendrocyte precursor cells in neonatal mice resulted in impaired cholesterol biosynthesis and defective myelinogenesis without compromising their differentiation into Aspa(+)Gstpi(+) myelinating oligodendrocytes. Mechanistically, Qki-5 functions as a co-activator of Srebp2 to control transcription of the genes involved in cholesterol biosynthesis in oligodendrocytes. Consequently, Qki depletion led to substantially reduced concentration of cholesterol in mouse brain, impairing proper myelin assembly. Our study demonstrated that Qki-Srebp2-controlled cholesterol biosynthesis is indispensable for myelinogenesis and highlights a novel function of Qki as a transcriptional co-activator beyond its canonical function as an RNA-binding protein.