We previously reported that paired-pulse activation of rat cerebellar granule cell (GC) ascending axon fibers at short intervals (30-100 ms) causes not only facilitation of the peak amplitude (PPF_amp) of the second EPSC (EPSC2) recorded from molecular layer interneurons (MLIs) but also prolongation of the EPSC2 decay time (PPP_decay) relative to those of the first EPSC (EPSC1). PPF_amp is the result of transient increases in release probability and multivesicular release (MVR). In contrast, PPP_decay is elicited by an increase in MVR and the subsequent pooling of MVR glutamate among adjacent active synapses as well as by a delayed release. We here report that (i) Ca_v2.1 and Ca_v2.2 channels differentially regulate PPP_decay and PPF_amp and (ii) the topographical distance between Ca_v2.1 channels and exocytotic Ca²⁺ sensors plays an important role in eliciting MVR. The amplitude of GC-MLI EPSCs decreased after application of the membrane-permeable slow Ca²⁺ chelator EGTA-AM. This decrease was stronger for EPSC2, thereby reducing PPF_amp significantly. In addition, EGTA-AM reduced τ_decay of EPSC2, resulting in the suppression of PPP_decay. It is therefore probable that Ca²⁺ sensors that mediate MVR are located more distally from Ca_v2.1 channels and Ca²⁺ accumulates even at release sites distant from the open channel. We further compared the effect of subtype-selective Ca_v2 channel blockers on GC-MLI EPSCs after treatment with EGTA-AM. ω-Agatoxin IVA and ω-conotoxin GVIA similarly reduced the amplitude of EPSC1 with a clear enhancement of PPF_amp. These Ca_v2 blockers did not cause any significant changes in PPP_decay after EGTA-AM treatment. Thus, Ca_v2.1 and Ca_v2.2 channels appeared to have similar impacts on transmitter release at the GC ascending axons when the accumulation of free Ca²⁺ and the following activation of distally located Ca²⁺ sensors (i.e., microdomain signaling) were suppressed.