In the mammalian brain, nerve cells exhibit large amplitude subthreshold membrane potential changes at their soma and dendrites. Here, we show by direct recordings (34 °C) from hippocampal granule cells and presynaptic mossy fiber boutons (MFBs) of the granule cell axon in combination with compartemental modelling that EPSPs are forward propagated (up to 0.9 mm) to the presynaptic structure in a decremental manner. To study the functional significance of these 'Excitatory Presynaptic Potentials' (EPreSPs), we performed paired-recordings at the MFB-CA3 pyramidal neuron synapse. The combination of EPreSPs and action potentials (APs) enhanced synaptic transmission to 1.43 ± 0.09 (n = 11) of control (AP only). Theta-like presynaptic membrane potential oscillations (5Hz) in combination with APs also enhanced synaptic transmission. In the presence of presynaptic 10 mM EGTA, a calcium chelator, a reduced but significant EPreSP enhancement remained (1.21 ± 0.04, n = 5). In addition, neither the presynaptic AP waveform nor the AP evoked presynaptic calcium current was affected by EPreSPs. Therefore, our data are consistent with a partial direct voltage modulation of release machinery underlying enhancement of synaptic transmission by presynaptic subthreshold membrane potential changes at this synapse.