Objective To define the functional significance of increased miR‐155 expression in myeloid cells in multiple sclerosis (MS). Methods miR‐155 expression levels were measured in CD14+ monocytes from untreated relapsing–remitting MS patients and compared to healthy controls. Similar microRNA (miRNA) analyses were performed in laser‐captured CD68+ cells from perivascular (blood‐derived macrophages) and parenchymal (microglia) brain regions in both active MS lesions and noninflammatory cases. Using human adult blood‐derived macrophages and brain‐derived microglia, in vitro experiments were performed to demonstrate how miR‐155 influences the polarization state, phenotype, and functional properties of myeloid cells, in addition to their ability to subsequently impact adaptive T‐cell responses. Results In MS, miR‐155 expression was significantly increased in both peripheral circulating CD14+ monocytes and active lesions (CD68+ cells) compared to control donor monocytes and parenchymal microglia, respectively. In vitro, miR‐155 was significantly increased in both M1‐polarized primary human macrophages and microglia. Transfection of an miR‐155 mimic increased proinflammatory cytokine secretion and costimulatory surface marker expression in both cell types; an miR‐155 inhibitor decreased proinflammatory cytokine expression. Coculture experiments demonstrated that allogeneic T‐cell responses were significantly enhanced in the presence of miR‐155–transfected myeloid cells compared to controls. Interpretation Our results demonstrate that miR‐155 regulates proinflammatory responses in both blood‐derived and central nervous system (CNS)‐resident myeloid cells, in addition to impacting subsequent adaptive immune responses. Differential miRNA expression may therefore provide insight into mechanisms responsible for distinct phenotypic and functional properties of myeloid cells, thus impacting their ability to influence CNS injury and repair. Ann Neurol 2013;74:709–720