INTRODUCTION: Slow transit constipation (STC) is a type of functional constipation. The detailed mechanism of STC, for which there is currently no effective treatment, is unknown as of yet. Tongbian decoction (TBD), a traditional Chinese medicinal formula, is commonly used to treat STC in clinical settings. However, the potential impact of TBD on the management of STC via modulation of the gut microbiota remains unclear. METHODS: Pseudo-germ-free rats were constructed after 6 days of treatment with bacitracin, neomycin, and streptomycin (abbreviated as ABX forthwith). Based on the successful construction of pseudo-germ-free rats, the STC model (ABX + STC) was induced using loperamide hydrochloride. After successful modeling, based on the different sources of donor rat microbiota, the ABX + STC rats were randomly divided into three groups: Control → ABX + STC, STC → ABX + STC, and STC + TBD → ABX + STC for fecal microbiota transplant (FMT). Body weight, fecal water content, and charcoal power propelling rate of the rats were recorded. Intestinal microbiota was detected by 16S rRNA sequencing, and the 5-hydroxytryptamine (5-HT) signaling pathway was examined by western blots, immunofluorescence, and immunohistochemical analysis. RESULTS: After treatment with fecal bacterial solutions derived from rats treated with Tongbian decoction (TBD), there was an increase in body weight, fecal water content, and the rate of charcoal propulsion in the rats. Additionally, activation of the 5-hydroxytryptamine (5-HT) signaling pathway was observed. The 16S rRNA sequencing results showed that the fecal bacterial solution from TBD-treated rats affected the intestinal microbiota of STC rats by increasing the proliferation of beneficial bacteria and suppressing the expansion of harmful bacteria. CONCLUSION: Our study showed that TBD alleviated constipation in STC rats by modulating the structure of the intestinal microbiota.