Oxytocin (OT) is a developmentally important neuropeptide recognised to play a dominant role in social functioning and stress-related behaviours, in a sex-dependent manner. Nonetheless, the underlining factors driving OT and OT receptor (OTR) early brain development remain unclear. Recent evidence highlight the critical influence of gut microbiota and its bidirectional interaction with the brain on neurodevelopment via the gut microbiota-brain axis. Therefore, we aimed to determine the impact of gut microbiota on the OTR system of the rat brain at different developmental stages in a pilot study. Quantitative OTR [125 I]-OVTA autoradiographic binding was carried out in the forebrain of male and female conventional (CON) and germ-free (GF) rats at postnatal days (PND) 8, 22 and 116-150. OTR binding was also assessed in the eyes of PND 1 and PND 4 GF female rats. Significant 'microbiota x sex x region' interaction and age-dependent effects on OTR binding were demonstrated. Microbiota status influenced OTR levels in males but not females with higher levels of OTR observed in GF vs CON rats in the cingulate, prelimbic and lateral/medial/ventral orbital cortex, and septum across all age groups, while sex differences were observed in GF, but not in CON rats. Interestingly, OTRs present in the eyes of CON rats were abolished in GF rats. This is the first study to uncover a sex-specific role of gut microbiota on the central OTR system, which may have implications in understanding the developmental neuroadaptations critical for behavioural regulation and the aetiology of certain neurodevelopmental disorders.