--- - |2+ Key Points This study characterizes the mechanisms underlying defects in synaptic transmission when DRP1 is genetically eliminated. Viral‐mediated knockout of DRP1 from the presynaptic terminal at the mouse calyx of Held increased initial release probability, reduced the size of synaptic vesicle recycling pool, and impaired synaptic vesicle recycling. Transmission defects could be partially restored by increasing intracellular calcium buffering capacity with EGTA‐AM, implying close coupling of Ca2+‐channels to SVs was compromised. Acute restoration of ATP to physiological levels in the presynaptic terminal did not revert synaptic defects. Loss of DRP1 impairs mitochondrial morphology in the presynaptic terminal, which in turn seems to arrest synaptic maturation. Abstract Impaired mitochondrial biogenesis and function is implicated in many neurodegenerative diseases, and likely affects synaptic neurotransmission prior to cellular loss. Dynamin‐related protein 1 (DRP1) is essential for mitochondrial fission and is disrupted in neurodegenerative disease. In this study, we used the mouse calyx of Held synapse as a model to investigate the impact of presynaptic DRP1 loss on synaptic vesicle (SV) recycling and sustained neurotransmission. In vivo viral expression of CRE in ventral cochlear neurons of floxed‐DRP1 mice generated a presynaptic‐specific DRP1‐KO (DRP1‐preKO), where the innervated postsynaptic cell was unperturbed. Confocal reconstruction of the calyx terminal suggests SV clusters and mitochondrial content are disrupted, and presynaptic terminal volume was decreased. Using postsynaptic voltage‐clamp recordings, we find that DRP1‐preKO synapses had larger evoked responses at low frequency stimulation. DRP1‐preKO synapses also had profoundly altered short‐term plasticity, due to defects in SV recycling. Readily‐releasable pool (RRP) size, estimated with high‐frequency trains, was dramatically reduced in DRP1‐preKO, suggesting an important role for DRP1 in maintenance of release competent SVs at the presynaptic terminal. Presynaptic Ca2+ accumulation in the terminal was also enhanced in DRP1‐preKO. Synaptic transmission defects could be partially rescued with EGTA‐AM, indicating close coupling of Ca channel to SV distance normally found in mature terminals may be compromised by DRP1‐preKO. Using paired recordings of the presynaptic and postsynaptic compartments, recycling defects could not be reversed by acute dialysis of ATP into the calyx terminals. Taken together, our results implicate a requirement for mitochondrial fission to coordinate postnatal synapse maturation. This article is protected by copyright. All rights reserved - The Journal of Physiology, Volume 0, Issue ja, -Not available-.