Spontaneous 'mini' release occurs at all synapses, but its nature remains enigmatic. We found that >95% of spontaneous release in murine cortical neurons was induced by Ca²⁺-binding to synaptotagmin-1 (Syt1), the Ca²⁺ sensor for fast synchronous neurotransmitter release. Thus, spontaneous and evoked release used the same Ca²⁺-dependent release mechanism. As a consequence, Syt1 mutations that altered its Ca²⁺ affinity altered spontaneous and evoked release correspondingly. Paradoxically, Syt1 deletions (as opposed to point mutations) massively increased spontaneous release. This increased spontaneous release remained Ca²⁺ dependent but was activated at lower Ca²⁺ concentrations and with a lower Ca²⁺ cooperativity than synaptotagmin-driven spontaneous release. Thus, in addition to serving as a Ca²⁺ sensor for spontaneous and evoked release, Syt1 clamped a second, more sensitive Ca²⁺ sensor for spontaneous release that resembles the Ca²⁺ sensor for evoked asynchronous release. These data suggest that Syt1 controls both evoked and spontaneous release at a synapse as a simultaneous Ca²⁺-dependent activator and clamp of exocytosis.