Importance Autoantibodies targeting astrocytes, such as those against glial fibrillary acidic protein (GFAP) or aquaporin protein 4, are crucial diagnostic markers for autoimmune astrocytopathy among central nervous system (CNS) autoimmune disorders. However, diagnosis remains challenging for patients lacking specific autoantibodies. Objective To characterize a syndrome of unknown meningoencephalomyelitis associated with an astrocytic autoantibody. Design, Setting, and Participants This retrospective case series study included samples collected from April 2021 to May 2024 at a tertiary referral hospital among patients with uncharacterized CNS autoimmune disorders and similar clinical and radiological features. Single-cell RNA sequencing (scRNA-seq) was performed on cerebrospinal fluid (CSF) cells of 2 index patients to identify the putative target antigen of the clonally expanded B cells. A comprehensive screening for additional patients was conducted using blinded cell-based and tissue-based assay. Candidate patients were followed up for a median (range) duration of 23 (5-31) months. Exposures scRNA-seq, autoantibody characterization, and testing. Main Outcomes and Measures Detection of the autoantibody and characterization of the associated autoimmune meningoencephalomyelitis. Results Fourteen candidate patients (10 [71%] female; median [IQR] age, 33 [23-41] years) were identified. Initially, CSF from 2 female patients with unknown encephalomyelitis showed astrocytic reactivity on rat tissue but was negative for GFAP IgG. A total of 17 of 37 clonally expanded B cell clonotypes (46%) in their CSF expressed IgG autoantibodies targeting the astrocytic intermediate filament protein vimentin. Subsequent screening identified 12 additional patients. These 14 patients shared a unique clinical profile characterized by relapsing courses and symptoms prominently involving the cerebellum, brainstem, and corticospinal tract (CST). All patients also exhibited elevated CSF protein and cells, intrathecal immunoglobulin synthesis, and magnetic resonance imaging (MRI) showing bilateral lesions on CST. Notably, 8 of 12 patients (67%) who received first-line immunotherapy at their first episode responded well. At the last follow-up, 11 patients (79%) experienced significant disability (modified Rankin Scale >= 3). Conclusions and Relevance In this case series, autoantibodies targeting the astrocytic intermediate filament protein vimentin were identified in patients with previously undifferentiated meningoencephalomyelitis and common radiographic features.