In all synapses, Ca²⁺ triggers neurotransmitter release to initiate signal transmission. Ca²⁺ presumably acts by activating synaptic Ca²⁺ sensors, but the nature of these sensors—which are the gatekeepers to neurotransmission—remains unclear. One of the candidate Ca²⁺ sensors in release is the synaptic Ca²⁺-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca²⁺ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca²⁺ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca²⁺ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.