Besides its role in triggering fast neurotransmitter release, the protein Synaptotagmin-1 (Syt1) is also involved in the docking and priming process of synaptic vesicles (SVs) to the presynaptic active zone. Research indicates that Synaptotagmin-7 (Syt7) shares some functions with Syt1, but the specific mechanisms by which Syt7 exerts these functions are not well understood. To investigate the putative redundant role of Syt1/Syt7 in SV docking and priming we performed various experiments as high-pressure freezing and electron microscopy on hippocampal cultures, and electrophysiological recordings on excitatory hippocampal autaptic neurons from Syt1/7 double knockout and Syt7 knockout mice. Our findings demonstrate that, similar to Syt1, Syt7 contributes to docking of SVs. Additionally, both Syt1 and Syt7 redundantly support priming of synaptic vesicles and the suppression of spontaneous release, consistent with previous research. By performing lentiviral overexpression experiments of Syt7 mutants carrying charge-neutralizing and membrane-binding mutations located at the top-loops of its C2A and C2B domains we explore the underlying molecular mechanism of Syt7 functions in SV dynamics and neurotransmitter release. We showed that putative Ca2+ and phospholipid binding activity of Syt7 C2A, but not C2B domain, is required for priming and clamping spontaneous release. Interestingly, analysis of Ca2+-triggered release revealed that Syt7 acts as negative regulator of release probability through its C2A domain function, providing a mechanism of its proposed role as regulator of presynaptic short-term plasticity. These findings contribute to a better understanding of the distinct functions of synaptotagmin family members in tuning spontaneous and Ca2+-triggered neurotransmitter release.