Chronic inflammation is a key pathological hallmark of multiple sclerosis and suggests that resolution of inflammation, orchestrated by specialized pro-resolving lipid mediators, is impaired. Here, through targeted-metabololipidomics in peripheral blood of patients with multiple sclerosis, we revealed that each disease form was associated with distinct lipid mediator profiles that significantly correlated with disease severity. In particular, relapsing and progressive multiple sclerosis patients were associated with high eicosanoids levels, whereas the majority of pro-resolving lipid mediators were significantly reduced or below limits of detection and correlated with disease progression. Furthermore, we found impaired expression of several pro-resolving lipid mediators biosynthetic enzymes and receptors in blood-derived leukocytes of MS patients. Mechanistically, differentially expressed mediators like LXA4, LXB4, RvD1 and PD1 reduced multiple sclerosis-derived monocyte activation and cytokine production and inhibited inflammation-induced blood-brain barrier dysfunction and monocyte transendothelial migration. Altogether, these findings reveal peripheral defects in the resolution pathway in multiple sclerosis, suggesting pro-resolving lipid mediators as novel diagnostic biomarkers and potentially safe therapeutics.