Glatiramer acetate (GA; Copaxone), a disease-modifying therapy for multiple sclerosis (MS), promotes development of anti-inflammatory (M2, type II) monocytes that can direct differentiation of regulatory T cells. We investigated the innate immune signaling pathways that participate in GA-mediated M2 monocyte polarization.Monocytes were isolated from myeloid differentiation primary response gene 88 (MyD88)-deficient, Toll-IL-1 receptor domain-containing adaptor inducing interferon (IFN)-β (TRIF)-deficient, IFN-α/β receptor subunit 1 (IFNAR1)-deficient, and wild-type (WT) mice and human peripheral blood. GA-treated monocytes were stimulated with Toll-like receptor ligands, then evaluated for activation of kinases and transcription factors involved in innate immunity, and secretion of proinflammatory cytokines. GA-treated mice were evaluated for cytokine secretion and susceptibility to experimental autoimmune encephalomyelitis.GA-mediated inhibition of proinflammatory cytokine production by monocytes occurred independently of MyD88 and nuclear factor-κB, but was blocked by TRIF deficiency. Furthermore, GA did not provide clinical benefit in TRIF-deficient mice. GA inhibited activation of p38 mitogen-activated protein kinase, an upstream regulator of activating transcription factor (ATF)-2, and c-Jun N-terminal kinase 1, which regulates IFN regulatory factor 3 (IRF3). Consequently, nuclear translocation of ATF-2 and IRF3, components of the IFN-β enhanceosome, was impaired. Consistent with these observations, GA inhibited production of IFN-β in vivo in WT mice, but did not modulate proinflammatory cytokine production by monocytes from IFNAR1-deficient mice.Our results demonstrate that GA inhibits the type I IFN pathway in M2 polarization of monocytes independently of MyD88, providing an important mechanism connecting innate and adaptive immune modulation in GA therapy and valuable insight regarding its potential use with other MS treatments.