The coupling of voltage-gated calcium channels (VGCCs) and vesicular calcium sensors for neurotransmitter release is crucial for fast and efficient synaptic transmission. At parallel fiber (PF)-Purkinje cell (PC) synapses in mouse cerebellum, the coupling tightens during synaptic maturation in parallel with switching of splicing isoforms of the P/Q-type VGCC (Cav2.1) EF-hand from EFb to EFa. The Cav2.1[EFa] isoform has been reported to have a tighter Ca2+ influx-sensor coupling distance than the Cav2.1[EFb] isoform in hippocampal pyramidal neurons. Thus, the shift in the splicing Cav2.1-EF hand isoforms may be involved in tightening the coupling at PF synapses. To address this hypothesis, we investigated the effect of splicing CaV2.1 EF-hand isoforms on the coupling distance in PF-PC synapses by the comparison of generated knock-in mice expressing only CaV2.1[EFb] with wildtype mice that predominantly express CaV2.1[EFa] using electrophysiology and electron microscopic technique called SDS-digested freeze-fracture replica labeling. A slow Ca2+ chelator EGTA significantly attenuated neurotransmitter release stronger, indicating a looser coupling, in EFb than in WT mice. Double labeling of CaV2.1 and Munc13-1, a marker of the SV docking site, by the freeze-fracture replica labeling revealed that the mean nearest neighbor distance from Munc13-1 to CaV2.1 was 25% farther at the PF active zone in EFb than in WT mice. These results suggest that the shift of alternative splicing Cav2.1 EF-hand isoforms at PF synapses causes a developmental tightening of Ca2+ influx-release coupling, contributing to fast and highly efficient neurotransmitter release.