Highly regulated and precise positioning of Ca2+ channels at the active zone (AZ) controls Ca2+ nanodomains at release sites. Their exact localization affects vesicular release probability (PVR) and is important for proper synaptic transmission during repetitive stimulation. We provide a detailed analysis of synaptic transmission combined with superresolution imaging of the AZ organization in mouse hippocampal synapses lacking Rab-interacting molecule-binding protein 2 (RIM-BP2). By dual- and triple-channel time-gated stimulated emission depletion (gSTED) microscopy, we directly show that RIM-BP2 fine-tunes voltage-gated Ca2+ channel 2.1 (CaV2.1) localization at the AZ. We reveal that RIM-BP2 likely regulates the Ca2+ nanodomain by positioning CaV2.1 channels close to synaptic vesicle release sites. Loss of RIM-BP2 reduces PVR and alters short-term plasticity.