Background: The gut microbiota (GM) plays a critical role in systemic health, influencing immune, metabolic, and neurological functions. Emerging evidence suggests that moxibustion, a traditional thermal therapy, may modulate GM to restore microbial homeostasis, yet its exclusive effects remain undifferentiated from combined therapies like acupuncture. Prior meta-analyses lack mechanistic specificity, necessitating a focused evaluation of moxibustion’s impact on microbial ecology. Objective: This systematic review and meta-analysis protocol aims to quantify moxibustion-induced changes in GM diversity, taxonomic composition, and functional metabolites (e.g., short-chain fatty acids [SCFAs]). Methods: We will systematically search PubMed, Web of Science, Cochrane Library, CNKI, Wanfang, and VIP databases from inception through December 31, 2024, using keywords such as “moxibustion,” “gut microbiota,” and “intestinal flora.” Eligible preclinical (animal) and clinical (human) studies evaluating standalone moxibustion interventions on gut microbiota will be included. Primary outcomes include microbial α-diversity indices (Shannon, Simpson) and relative abundance of key taxa (e.g., Firmicutes, Bacteroidetes). Risk of bias will be assessed using SYRCLE for animal studies and modified CAMARADES criteria for human trials. Pooled effect estimates for continuous outcomes (e.g., diversity indices, taxa ratios) will be calculated using the ratio of means (ROM) with 95% confidence intervals. Statistical analyses will be conducted in RevMan 5.4 and R (metafor package), with data archived on Figshare for reproducibility. Results: As of March 2025, the literature search and screening have been completed, and 31 studies meeting the inclusion criteria have been identified. The comprehensive analysis is scheduled to be completed by October 2025, with results anticipated to be published in late 2025. The anticipated findings suggest that moxibustion may reduce pathogenic genera such as Ruminococcus while enhancing beneficial genera, effects that are expected to be associated with improved intestinal barrier integrity and anti-inflammatory responses. Conclusions: This protocol provides a rigorous framework to evaluate moxibustion’s unique role in GM modulation, bridging traditional medicine with microbiome science. Results will inform optimized, non-pharmacological strategies for managing microbiome-associated chronic diseases and guide future research priorities. Clinical Trial: Prospero CRD42025639714.