The gut microbiome has emerged as a promising target for modulating adverse effects of opioid exposure due to its significant role in health and disease. Opioid use disorder (OUD) has become increasingly prevalent, specifically in women of reproductive age, contributing to an increased incidence of offspring exposed to opioids in utero. Recent studies have shown that prenatal opioid exposure (POE) is associated with notable changes to the maternal gut microbiome, with subsequent implications for the offspring's microbiome and other adverse outcomes. However, the role of the gut microbiome in mediating sex-based differences in pain sensitivity has not yet been investigated. In this study, both male and female C57BL/6 offspring were used to determine sex-based differences in nociception and gut microbial composition as a result of POE. Our data reveals significant sex-based differences in offspring prenatally exposed to opioids. The gut microbiome of opioid-exposed females showed an enrichment of commensal bacteria including compared to opioid-exposed males. Additionally, POE females demonstrated decreased nociceptive sensitivity, while males demonstrated increased nociceptive sensitivity. RNA sequencing of the prefrontal cortex showed sex-based differences in several canonical pathways, including an increase in the opioid signaling pathway of opioid-exposed females, which was not observed in males. Microbiome modification via maternal probiotic supplementation attenuated sex-based differences throughout the early stages of life. Together, our study provides further insight on sex-based differences arising from POE and highlights the pivotal role of the gut microbiome as a modifiable target for mitigating its negative effects.