Background: Screening of putative autoimmune targets in multiple sclerosis (MS) revealed a proportion of patients carrying antibodies (Abs) against KIR4.1, a potassium channel that shares functional properties with AQP4. Both are localized at the perivascular astrocytic processes. Aims: To measure anti-KIR4.1 Abs in the serum of MS and neuromyelitis optica (NMO) patients, and to identify the clinical and laboratory characteristics of patients harboring anti-KIR4.1 Abs. Methods: We measured anti-KIR4.1 Abs in serum, using the peptide KIR4.1 (83–120) enzyme-linked immunosorbent assay (ELISA). Results: Serum levels of anti-KIR4.1 Abs were significantly higher in MS and NMO patients than in healthy controls (HCs); with Abs detected in 21 of 80, 10 of 45, and 2 of 32 individuals, respectively (MS versus HC, p < 0.05). The level of anti-KIR4.1 Abs was significantly higher during MS relapse, versus remission ( p = 0.04). The clinical characteristics of our study patients did not vary based on KIR4.1 positivity. Conclusions: Anti-KIR4.1 Abs were found in similar proportions of patients with MS and NMO, at a significantly higher level than observed in HCs; consequently, the presence of Abs does not discriminate between these demyelinating diseases. However, anti-KIR4.1 Ab levels differed in MS patients during relapse and remission; as such, they may represent a marker of disease exacerbation.