Astrocytes constantly adapt their ramified morphology in order to support brain cell assemblies. Such plasticity is partly mediated by ion and water fluxes, which rely on the water channel aquaporin-4 (AQP4). The mechanism by which this channel locally contributes to process dynamics has remained elusive. Using a combination of single-molecule and calcium imaging approaches, we here investigated in hippocampal astrocytes the dynamic distribution of the AQP4 isoforms M1 and M23. Surface AQP4-M1 formed small aggregates that contrast with the large AQP4-M23 clusters that are enriched near glutamatergic synapses. Strikingly, stabilizing surface AQP4-M23 tuned the motility of astrocyte processes and favors glutamate synapse activity. Furthermore, human autoantibodies directed against AQP4 from neuromyelitis optica (NMO) patients impaired AQP4-M23 dynamic distribution and, consequently, astrocyte process and synaptic activity. Collectively, it emerges that the membrane dynamics of AQP4 isoform regulate brain cell assemblies in health and autoimmune brain disease targeting AQP4. Display omitted •AQP4 subtypes have different membrane trafficking and distribution in astrocytes•AQP4-M23 membrane dynamics are regulated by neuronal activity•AQP4-M23 membrane dynamics tune astrocyte process motility and synaptic transmission•Autoantibodies from NMO patients disturb AQP4 surface dynamics and related functions Ciappelloni et al. use fluorescent imaging approaches to investigate how astrocytes control their highly ramified processes. The membrane dynamics and distribution of aquaporin-4 (AQP4) subtypes control the motility of astrocyte processes, influencing the physiological interplay between astrocyte and neurons. This regulation is targeted by autoantibodies from NMO patients.