Statins have attracted interest as a treatment option for multiple sclerosis (MS) because of their pleiotropic antiinflammatory and immunomodulatory effects. However, contradictory results have been described when they are applied to oligodendrocytes (OLGs), the cell type predominantly affected in MS. In this study we focus on the in vitro effect of statins on process outgrowth in OLN-93 cells, a well-characterized OLG-derived cell line, and primary cultures of neonatal rat OLGs. Application of the lipophilic simvastatin, as low as 0.1-1 μM, disturbs process formation of both cell types, leading to less ramified cells. We show that both protein isoprenylation and cholesterol synthesis are required for the normal differentiation of OLGs. It is further demonstrated that the expression of 2',3'-cyclic-nucleotide-3' phosphodiesterase (CNP) and tubulin is lowered, concomitant with a reduction of membrane-bound CNP as well as tubulin. Therefore, we propose that lack of isoprenylation of CNP could help to explain the altered morphological and biochemical differentiation state of treated OLGs. Moreover, expression of specific myelin markers, such as myelin basic protein, myelin-associated glycoprotein, and myelin oligodendrocyte glycoprotein, was compromised after treatment. We conclude that simvastatin treatment has detrimental effects on OLG process outgrowth, the prior step in (re)myelination, thereby mortgaging long-term healing of MS lesions.