Signal transmission between neurons requires exocytosis of neurotransmitters from the lumen of synaptic vesicles into the synaptic cleft. Following an influx of Ca2+, this process is facilitated by the Ca2+ sensor synaptotagmin-1. The underlying mechanisms involve electrostatic and hydrophobic interactions tuning the lipid preferences of the two C2 domains of synaptotagmin-1; however, the details are still controversially discussed. We, therefore, follow a multidisciplinary approach and characterize lipid and membrane binding of the isolated C2A and C2B domains. We first target interactions with individual lipid species, and then study interactions with model membranes of liposomes. Finally, we perform molecular dynamics simulations to unravel differences in membrane binding. We found that both C2 domains, as a response to Ca2+, insert into the lipid membrane; however, C2A adopts a more perpendicular orientation while C2B remains parallel. These findings allow us to propose a mechanism for synaptotagmin-1 during membrane fusion.