At birth, the auditory system of most mammals is functionally immature and requires a postnatal period of synaptic refinement to achieve the precise connectivity observed in adulthood. During this developmental window, the medial olivocochlear (MOC) efferent system modulates spontaneous inner ear activity, thereby affecting the maturation of central circuits. In mice with enhanced MOC activity (α9KI), synaptic dysfunction was found at the calyx of Held (CH) within the medial nucleus of the trapezoid body (MNTB) (Di Guilmi et al., 2019). In this study, we combined in vitro electrophysiology (P12–14) with 3D morphological reconstructions (P12–14 and P21–25) using serial electron microscopy in three genotypes: wild type (WT), α9KI, and α9KO (lacking MOC activity) and a custom Python-based code to extract quantitative parameters, enabling the classification of CHs into distinct morphotypes. Recordings in the α9KI mice displayed synaptic alterations across several parameters, evidenced by a lower excitatory post-synaptic current, a higher short-term depression and smaller ready realizable vesicle pool. Morphological analyses revealed a lower proportion of structurally complex CHs and decreased synaptic pruning in α9KI animals. By contrast, α9KO only showed reduced morphological complexity. These results suggest that enhanced MOC activity drives more profound developmental modifications in MNTB circuitry compared to the absence of MOC modulation.