We have studied the effects of one and two Synaptotagmin-1 C2B domains along the expansion of the fusion pore, with the following conclusions: (i) one and two C2B domains in the cytosolic space cumulatively reduce the energetic expansion cost, (ii) one C2B domain marginally increases the nucleation-expansion barrier while two C2B domains significantly reduce it, and (iii) C2B domains systematically push just nucleated nascent fusion pores toward expansion.