Neurotransmitter release is regulated by several proteins localized at the active zones of presynaptic terminals. Munc13-1 is a multi-domain active zone protein which redundantly interacts with other active zone proteins including RIM. We have previously shown that Munc13-1 forms supramolecular nanoassemblies that function as the synaptic vesicle release sites. Here, we aimed to clarify a more detailed molecular mechanism of the formation/maintenance of Munc13-1 nanoassemblies by intervening in the interaction of Munc13-1 with RIM. For this purpose, we isolated the Zn²⁺ finger domain of RIM (RIM-ZF), which interacts with the C₂A domain of Munc13-1 and expressed it in neurons to competitively inhibit the binding of endogenous Munc13-1 and RIM. Glutamate imaging revealed that the expression of RIM-ZF in cultured neurons caused a significant decrease in neurotransmitter release, although a previous study showed that the RIM-ZF partially rescues suppressed neurotransmitter release in RIM knock-out neurons. Furthermore, quantitative immunocytochemical analysis with super-resolution microscopy revealed that the expression of RIM-ZF altered the nanoscale distribution of Munc13-1 molecules at the active zones. Thus, our results suggest that a direct interaction of RIM-ZF with Munc13-1 itself is incomplete for the appropriate formation of synaptic vesicle release sites, and that cross-linkage of RIM-ZF to the other domains of RIM is necessary for the precise positioning of Munc13-1 at the active zone.