Purpose: To investigate levels of oxysterols in healthy control (HC) and multiple sclerosis (MS) patients and their interdependence with demographic, clinical characteristics, and cholesterol biomarkers. Methods: This study included 550 subjects (203 HC, 221 relapsing-remitting MS (RR-MS), 126 progressive MS (P-MS)). A complete lipid profile including total cholesterol (TC); high-density lipoprotein-cholesterol (HDL-C); low-density lipoprotein-cholesterol (LDL-C); apolipoproteins (Apo) A1, A2, B, and E; C-reactive protein (CRP); 24-hydroxycholesterol (HC); 25-HC; 27-HC; 7-HC; and 7-ketocholesterol (KC) was obtained. Lipoprotein particle sizing by proton nuclear magnetic resonance (H-1 NMR) was available for 432 subjects. Results: The levels of 24-HC, 27-HC, and 7-HC (all p<0.015) were lower in MS compared to HC, and 7-KC was higher in P-MS compared to RR-MS (p<0.001). TC, LDL-C, and ApoB were associated with higher levels of all oxysterols (all p<0.05) in HC. In MS, LDL-C was associated with higher levels of 24-HC, 25-HC, 7-KC, and 7-HC (all p<0.05), while TC and ApoB were associated with increased levels of all oxysterols (all p<0.005). Conclusion: The findings of lower 24-HC, 27-HC, and 7-HC in MS compared to HC and higher 7-KC in P-MS compared to RR-MS indicate that the oxysterol network is disrupted in MS.