Our previous study showed that 3-day forced swimming (FS) stress induced long-lasting comorbid visceral pain and referred hyperalgesia in the low back area of female rats with chronic constriction injury of the infraorbital nerve (CCI), which was mediated by 5-HT-dependent descending projection and selective upregulation of 5-HT3R in the sacral spinal cord. Recent studies suggest the involvement of the gut microbiota in stress-related and visceral pain conditions. However, whether changes in the gut microbiota contribute to comorbid visceral pain is unknown. Here, we determine that compositional and structural changes in the gut microbiota of rats with existing orofacial neuropathic pain contribute to stress-induced comorbid visceral pain. We conducted 16S rRNA gene amplicon sequence analysis using fecal samples. Our results indicate significant alterations in the gut microbiota of CCI-treated rats before FS stress, along with a substantial increase in the Firmicute to Bacteroidetes ratio, especially an increase in the abundance of 5-HT-stimulating gut bacteria, including Clostridiaceae, Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae. These changes resolved by 21 days following FS stress despite ongoing referred hyperalgesia in the low back. In contrast, the rats subjected to sham surgery or only FS stress did not exhibit significant alterations in the gut microbiota and did not present with referred hyperalgesia. Next, to determine whether the observed changes in the gut microbiota were necessary for comorbid visceral pain, we performed reciprocal fecal microbiota transfer (FMT) experiments. We found that significant changes in the gut microbiota of naïve recipients of FMT from orofacial pain donors were consistent with donor microbial profiles. In these FMT recipients, FS stress induced long-lasting referred hyperalgesia. FMT from naïve donors normalized the gut microbiota profiles of the rats with comorbid visceral pain and significantly attenuated their referred hyperalgesia. Finally, FMT experiments confirmed that the changes in the gut microbiota contributed to the increase in 5-HT in the colon and upregulation of 5-HT3R expression in the sacral DRGs and spinal dorsal horn in rats with comorbid visceral pain. These changes may mediate 5-HT-dependent descending facilitation and are involved in the peripheral mechanisms underlying stress-induced comorbid visceral pain.