Transmitter release at the hippocampal mossy fiber (MF)-CA3 synapse exhibits robust use-dependent short-term plasticity with an extremely wide dynamic range. Recent studies revealed that presynaptic kainate receptors (KARs), which specifically localized on the MF axons, mediate unusually large facilitation at this particular synapse in concert with the action of residual Ca2+. However, it is currently unclear how activation of kainate autoreceptors enhances transmitter release in an activity-dependent manner. Using fluorescence recordings of presynaptic Ca2+ and voltage in hippocampal slices, here we demonstrate that paired-pulse stimulation (with 20-200 msec intervals) resulted in facilitation of Ca2+ influx into the MF terminals, as opposed to other synapses, such as the Schaffer collateral-CA1 synapse. These observations deviate from typical residual Ca2+ hypothesis of facilitation, assuming an equal amount of Ca2+ influx per action potential. Pharmacological experiments reveal that the facilitation of presynaptic Ca2+ influx is mediated by activation of KARs. We also found that action potentials of MF axons are followed by prominent afterdepolarization, which is partly mediated by activation of KARs. Notably, the time course of the afterdepolarization approximates to that of the paired-pulse facilitation of Ca2+ influx, suggesting that these two processes are closely related to each other. These results suggest that the novel mechanism amplifying presynaptic Ca2+ influx may underlie the robust short-term synaptic plasticity at the MF-CA3 synapse in the hippocampus, and this process is mediated by KARs whose activation evokes prominent afterdepolarization of MF axons and thereby enhances action potential-driven Ca2+ influx into the presynaptic terminals.