Spreading depolarization (SD) is a slowly propagating wave of neuronal and glial depolarization. A growing number of studies show that SD and SD-like phenomena play a role in neurological disorders such as migraine, stroke, and traumatic brain injury. Despite the clinical importance of SD, its underlying molecular and cellular mechanisms remain elusive, possibly because of insufficient animal model allowing genetic manipulation. Such a model would also allow high-throughput screening for SD-suppressing drug development. To address this, we developed a novel experimental system to study SD using zebrafish. Electrophysiological recordings in the immobilized adult zebrafish revealed that increasing extracellular potassium concentration elicited SD with a large and long-lasting negative shift of direct current (DC) potential in the optic tectum. It also reduced the oscillatory activity in the extracellular field potential and increased the expression of the immediate early gene c-fos. Pharmacological blocking of the N-methyl-d-aspartate (NMDA) glutamate receptor attenuated the propagation of SD, suggesting that glutamatergic neurotransmission mediated tectal SD in zebrafish. Our analyses revealed that the zebrafish tectum and rodent cortex had similar SD kinetics. The current study provides electrophysiological and pharmacological evidence that zebrafish SD and mammal SD are comparable. This zebrafish SD model is suitable for genetic manipulation and cost-effective high-throughput screening. It could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.NEW & NOTEWORTHY Previous studies have implicated spreading depolarization (SD) in stroke and migraine. Here, we demonstrate SD, for the first time, in the adult zebrafish tectum showing waveform kinetics, c-fos expression, and attenuation by N-methyl-d-aspartate glutamate receptor blocker as observed in the rodent cortex. Since the zebrafish is an animal model amenable to genetic manipulation and chemical screening, this result could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.