Synaptic transmission is a complex process, which requires activation of presynaptic Ca2+ channels, Ca2+ influx and binding to release sensors. Synaptotagmin 1 (Syt-1) has been previously identified as the primary Ca2+ sensor for fast synchronous transmitter release, while synaptotagmin 7 (Syt-7) was suggested to mediate slower, asynchronous release. However, recent reports have conflicting conclusions on the mechanisms of vesicle release modes, making our understanding of Ca2+ sensing in transmission unclear. To understand the action of synaptotagmins at a central synapse, we performed subcellular paired patch-clamp recordings between hippocampal mossy fiber boutons and CA3 pyramidal cells in acute slices from conditional Syt-1 (Syt-1fl/fl x Prox1-Cre) and constitutive Syt-7 knockout mice. Presynaptic terminals were stimulated non-invasively in the cell-attached configuration, allowing optimal characterization of synaptic properties. We found that Syt-1 deletion from dentate gyrus granule cells abolished stimulus-evoked synchronous EPSCs (232.4 pA control; 45.4 pA Syt-1 cKO; n = 12 pairs) and unclamped asynchronous release. Despite this effect, Syt-1 cKO synapses were still able to trigger spiking in postsynaptic CA3 pyramidal cells. However, while the conditional detonator properties of the synapse were maintained, the timing precision of postsynaptic spikes was reduced. Contrary to previous findings, ablation of Syt-7 had only minimal effects on synaptic transmission; both vesicle pool dynamics and synaptic facilitation remained largely unaffected. Together, our results identify Syt-1 as the main Ca2+ sensor at the hippocampal mossy fiber synapse and demonstrate that Syt-1 plays a key role in regulating the timing of synaptic signaling in the hippocampal trisynaptic circuit.