Phosphorylation and dephosphorylation are primary means for rapid regulation of a variety of neuronal functions, such as membrane excitability, neurotransmitter release, and gene expression. Voltage-gated Ca²⁺ channels are targets for phosphorylation by a variety of second messengers through activation of different types of protein kinases (PKs). Protein phosphatases (PPs), like PKs, are equally important in regulating Ca²⁺ channels in neurons. However, much less is understood about whether and how a particular type of PP contributes to regulating neuronal Ca²⁺ channel activities. This is primarily because of the lack of specific inhibitors/activators for different types of PPs, particularly the PP2c family. The functional roles of PP2c and its substrates in the brain remain virtually unknown. During our yeast two-hybrid screening, PP2cα was pulled out by both N- and P/Q-type Ca²⁺ channel C termini. This raised the possibility that PP2cα might be associated with voltage-gated Ca²⁺ channels for regulation of the Ca²⁺ channel activity. Biochemical studies show that PP2cα binds directly to neuronal Ca²⁺ channels forming a functional protein complex in vivo. PP2cα, unlike PP1, PP2a and PP2b, is more effective in dephosphorylation of neuronal Ca²⁺ channels after their phosphorylation by PKC. In hippocampal neurons, disruption of the PP2cα-Ca²⁺ channel interaction significantly enhances the response of Ca²⁺ channels to modulation by PKC. Thus, the PP2cα-Ca²⁺ channel complex is responsible for rapid dephosphorylation of Ca²⁺ channels and may contribute to regulation of synaptic transmission in neurons.