μ-opioid receptors (MOPr) play a critical role in social play, reward and pain, in a sex- and age-dependent manner. There is evidence to suggest that sex and age differences in brain MOPr density may be responsible for this variability; however, little is known about the factors driving these differences in cerebral MOPr density. Emerging evidence highlights gut microbiota's critical influence and its bidirectional interaction with the brain on neurodevelopment. Therefore, we aimed to determine the impact of gut microbiota on MOPr density in male and female brains at different developmental stages. Quantitative [3H]DAMGO 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. Significant ‘microbiota status X sex’, ‘age X brain region’ interactions and microbiota status- and age-dependent effects on MOPr binding were uncovered. Microbiota status influenced MOPr levels in males but not females, with higher MOPr levels observed in GF versus CON rats overall regions and age groups. In contrast, no overall sex differences were observed in GF or CON rats. Interestingly, within-age planned comparison analysis conducted in frontal cortical and brain regions associated with reward revealed that this microbiota effect was restricted only to PND22 rats. Thus, this pilot study uncovers the critical sex-dependent role of gut microbiota in regulating cerebral MOPr density, which is restricted to the sensitive developmental period of weaning. This may have implications in understanding the importance of microbiota during early development on opioid signalling and associated behaviours.