Neuronal differentiation is accomplished through cascades of intrinsic genetic factors initiated in neuronal progenitors by external gradients of morphogens. Activity was thought to be important only late in development, but recent evidence indicates that activity also regulates early neuronal differentiation. Activity in post-mitotic neurons prior to synapse formation can regulate phenotypic specification, including neurotransmitter choice, but the mechanisms are not clear. Here we identify a mechanism that links endogenous calcium spike activity with an intrinsic genetic pathway to specify neurotransmitter choice in neurons in the dorsal embryonic spinal cord of Xenopus tropicalis. Early activity modulates transcription of the GABAergic/glutamatergic selection gene tlx3 and requires a variant cAMP response element (CRE) in its promoter. The cJun transcription factor binds to this CRE site, modulates transcription, and regulates neurotransmitter phenotype through its transactivation domain. Calcium signals through cJun N-terminal phosphorylation, thus integrating activity-dependent and intrinsic neurotransmitter specification. This mechanism provides a basis for early activity to regulate genetic pathways at critical decision points, switching the phenotype of developing neurons.