The β subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain β subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β4subunit lacking the variable N terminus (β4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length β4variants (β4aand β4b), β4eis most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of β4subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (β4-null) mice individually reconstituted with β4a, β4b, and β4e. Notably, the number of genes regulated by each β4splice variant correlated with the rank order of their nuclear-targeting properties (β4b> β4a> β4e). Together, these findings support isoform-specific functions of β4splice variants in neurons, with β4bplaying a dual role in channel modulation and gene regulation, whereas the newly detected β4evariant serves exclusively in calcium-channel-dependent functions.