Synaptotagmin-1 is the calcium sensor for rapid, synchronous neurotransmitter release, but how calcium binding leads to fusion is not understood. We tested the effects of Synaptotagmin's cytoplasmic C2 domains (C2AB) on fusion pore opening and dilation using a recently established single-pore conductance assay which probes fusion between nanodiscs reconstituted with neuronal v-SNAREs and engineered cells that express the complementary t-SNAREs in a flipped configuration on the cell surface. Fusion results in a nm-sized fusion pore that can be detected in cell-attached, voltage-clamped recordings. Compared to fusion induced by SNAREs alone, in the presence of C2AB the fusion rate, the mean single-pore conductance, and the pore lifetime increased, but only in the presence of calcium and PI(4,5)P2. The assay recapitulated effects of previously characterized mutations on Synaptotagmin. In particular, abolishing binding of Synaptotagmin to the SNARE complex or calcium via mutagenesis eliminated Synaptotagmin'ss effects on pore dilation. Binding of calcium to Synaptotagmin does not alter its interactions with the SNARE complex, suggesting rapid calcium-dependent Synaptotagmin-bilayer interactions may be key to triggering fusion. We probed interactions between single C2AB domains and lipid bilayers using high-resolution optical tweezers. It was recently shown that in the presence of calcium, Synaptotagmin-1 C2AB domains bound and unbound the membrane rapidly, releasing ∼8-13 kT per C2AB molecule, depending on the lipid composition. Here we show that disrupting binding of C2AB to PI(4,5)P2 through mutagenesis reduced the binding energy, while disrupting binding to calcium abolished C2AB-membrane interactions. Together, our results indicate that Synaptotagmin-1's effects on fusion pore opening and dilation require it to interact both with membranes and SNAREs. Additionally they suggest the role of Synaptotagmin-1 is not confined to triggering fusion pore opening; it also plays a post-fusion role in pore dilation in the absence of additional factors.