Due to its pivotal role in the modulation of immune and inflammatory responses, the gut microbiota has emerged as a key modulator of cancer treatment-induced gastrointestinal mucositis. However, it is not clear yet how it affects radiation therapy-induced oral mucositis (OM). As such, this study aimed to explore the gut microbiota's role in the pathogenesis of radiation-induced OM in rats. Male Sprague Dawley rats were treated with 20 Gy x-ray radiation (Rx) delivered to the snout, with or without antibiotic-induced microbiota depletion (AIMD). OM severity was assessed, and tongue tissues were collected on day 9 and 15 postradiation for tissue injury and inflammatory markers assessment. AIMD+Rx had a significantly shorter duration of severe OM compared with Rx alone group. Macroscopically, the tongue ulcer-like area was smaller in AIMD+Rx compared with the Rx group. Microscopically, a smaller percentage of the mucosal ulcer was observed in the dorsal tongue of AIMD+Rx compared with the Rx group. AIMD+Rx also had significantly lower levels of interleukin 6, interleukin 1 beta, and toll like receptor 4 in the tongue tissues than the Rx group. The gut microbiota plays a role in OM pathogenesis, mainly in the recovery phase, through the modulation of proinflammatory pathways. Future microbiota-targeted interventions may improve OM in clinical settings.