Axon specification is a critical step in neuronal development, and the function of glial cells in this process is not fully understood. Here, we show that GLR glial cells regulate axon specification of their nearby GABAergic RME neurons through GLR-RME gap junctions. Disruption of GLR-RME gap junctions causes misaccumulation of axonal markers in non-axonal neurites of RME neurons and converts microtubules in those neurites to form an axon-like assembly. We further uncover that GLR-RME gap junctions regulate RME axon specification through activation of the CDK-5 pathway in a calcium-dependent manner, involving a calpain . Therefore, our study reveals the function of glia-neuron gap junctions in neuronal axon specification and shows that calcium originated from glial cells can regulate neuronal intracellular pathways through gap junctions.