The calyx of Held is a giant nerve terminal mediating high-frequency excitatory input to principal cells of the medial nucleus of the trapezoid body (MNTB). MNTB principal neurons are enwrapped by densely organized extracellular matrix structures, known as perineuronal nets (PNNs). Emerging evidence indicates the importance of PNNs in synaptic transmission at the calyx of Held. Previously, a unique differential expression of aggrecan and brevican has been reported at this calyceal synapse. However, the role of hyaluronan and proteoglycan binding link proteins (HAPLNs) in PNN formation and synaptic transmission at this synapse remains elusive. This study aimed to assess immunohistochemical evidence for the effect of HAPLN4 on differential PNN formation at the calyx of Held. Genetic deletion of Hapln4 exhibited a clear ectopic shift of brevican localization from the perisynaptic space between the calyx of Held terminals and principal neurons to the neuropil surrounding the whole calyx of Held terminals. In contrast, aggrecan expression showed a consistent localization at the surrounding neuropil, together with HAPLN1 and tenascin-R, in both gene knockout (KO) and wild-type (WT) mice. An in situ proximity ligation assay demonstrated the molecular association of brevican with HAPLN4 in WT and HAPLN1 in gene KO mice. Further elucidation of the roles of HAPLN4 may highlight the developmental and physiological importance of PNN formation in the calyx of Held.