Significance Acetylcholine (ACh) produced by neurons performs an array of functions that control cardiac, gastrointestinal, and other biosystems. Here we discovered that lymphocytic natural killer (NK) cells bear machinery that produces ACh. The activity of ACh-producing NK cells up-regulates during the disease flare of multiple sclerosis (MS) and may, therefore, reflect the pathologic state. In the mouse model of MS, experimental autoimmune encephalomyelitis, these ACh-producing NK cells can reduce the intensity of inflammation and autoimmune responses in the brain and spinal cord. Therefore, the nonneural cholinergic system, as reflected by ACh-producing NK cells, appeared to counteract aberrant immune responses and lessen brain damage. This observation offers insight into the therapeutic mechanisms of the Food and Drug Administration-approved drug daclizumab high-yield process for MS.