Purpose <p>In the central nervous system, Ca²⁺ signaling plays a pivotal role in various cellular processes, including neuronal development and maturation. Disruption of Ca²⁺ homeostasis impairs auditory circuit formation, compromising auditory processing and behavior. Here, we aimed to investigate systematically the development of Ca²⁺ signaling in neurons of the medial nucleus of the trapezoid body (MNTB), a brainstem nucleus critical for sound localization.</p> Methods <p>Using Ca²⁺ imaging in brain slices from mice genetically expressing the Ca²⁺ indicator GCaMP in glycinergic cells, we studied Ca²⁺ signaling in MNTB neurons from mice of either sex at three developmental milestone stages, in response to both bath-applied agonists of various neurotransmitter receptors and synaptic activation of glutamatergic afferents.</p> Results <p>Prior to hearing onset at postnatal 7&#xa0;days (P7), robust Ca²⁺ responses were evoked in MNTB neurons upon activation of glutamate receptors (NMDARs, AMPARs, and group I (Gp-I) mGluRs by 200&#xa0;μM NMDA, 100&#xa0;μM AMPA, and 200&#xa0;μM 3,5-DHPG, respectively), whereas after hearing onset (P14 and P21), responses induced by NMDA and 3,5-DHPG declined markedly while AMPA-induced responses remained relatively strong. Ca²⁺ responses upon application of GABA (100&#xa0;μM) and glycine (200&#xa0;μM) were detected in neonatal mice, diminished in a few days after birth, and almost completely disappeared by P7. Whole-cell patch-clamp recordings showed that stimulation of excitatory afferents evoked action potentials across all ages with no differences in firing frequency up to 100&#xa0;Hz, but Ca²⁺ responses varied in a stimulus intensity- and frequency-dependent manner and exhibited developmental downregulation.</p> Conclusion <p>Ca²⁺ signaling induced by activation of the major transmitter receptors in MNTB neurons is highly developmentally down-regulated.</p>