The increasing use of mobile communication has triggered an interest in its possible effects on the regulation of neurotransmitter signals. Due to the close proximity of mobile phones to hearing-related brain regions during usage, its use may lead to a decrease in the ability to segregate sounds, leading to serious auditory dysfunction caused by the prolonged exposure to radiofrequency (RF) radiation. The interplay among auditory processing, excitation and inhibitory molecule interactions plays a major role in auditory function. In particular, inhibitory molecules, such a glycine, are predominantly localized in the auditory brainstem. However, the effects of exposure to RF radiation on auditory function have not been reported to date. Thus, the aim of the present study was to investigate the effects of exposure to RF radiation on glycine receptor (GlyR) immunoreactivity (IR) in the auditory brainstem region at 835 MHz with a specific absorption rate of 4.0 W/kg for three months using free-floating immunohistochemistry. Compared with the sham control (SC) group, a significant loss of staining intensity of neuropils and cells in the different subdivisions of the auditory brainstem regions was observed in the mice exposed to RF radiation (E4 group). A decrease in the number of GlyR immunoreactive cells was also noted in the cochlear nuclear complex [anteroventral cochlear nucleus (AVCN), 31.09%; dorsal cochlear nucleus (DCN), 14.08%; posteroventral cochlear nucleus (PVCN), 32.79%] and the superior olivary complex (SOC) [lateral superior olivary nucleus (LSO), 36.85%; superior paraolivary nucleus (SPN), 24.33%, medial superior olivary nucleus (MSO), 23.23%; medial nucleus of the trapezoid body (MNTB), 10.15%] of the mice in the E4 group. Auditory brainstem response (ABR) analysis also revealed a significant threshold elevation of in the exposed (E4) group, which may be associated with auditory dysfunction. The present study suggests that the auditory brainstem region is susceptible to chronic exposure to RF radiation, which may affect the function of the central auditory system.