Sublethal injurious stimuli in neurons induce transient increases in free intracellular Zn2+ that are associated with regulating adaptive responses to subsequent lethal injury, including alterations in the function and localization of the delayed-rectifier potassium channel, Kv2.1. However, the link between intracellular Zn2+ signalling and the observed changes in Kv2.1 remain undefined. In the present study, utilizing exogenous Zn2+ treatment, along with a selective Zn2+ ionophore, we show that transient elevations in intracellular Zn2+ concentrations are sufficient to induce calcineurin-dependent Kv2.1 channel dispersal in rat cortical neurons in vitro, which is accompanied by a relatively small but significant hyperpolarizing shift in the voltage-gated activation kinetics of the channel. Critically, using a molecularly encoded calcium sensor, we found that the calcineurin-dependent changes in Kv2.1 probably occur as a result of Zn2+-induced cytosolic Ca2+ release via activation of neuronal ryanodine receptors. Finally, we couple this mechanism with an established model for in vitro ischaemic preconditioning and show that Kv2.1 channel modulation in this process is also ryanodine receptor-sensitive. Our results strongly suggest that intracellular Zn2+-initiated signalling may represent an early and possibly widespread component of Ca2+-dependent processes in neurons.