Functional constipation (FC) is a common digestive disorder that affects patients’ quality of life and is closely associated with intestinal tumors. This study used a cross-sectional design to assess the changes of intestinal flora and metabolites in different severities of FC patients through 16S rRNA sequencing and metabolomics analysis. Results showed that patients with severe FC had significantly higher clinical and anxiety scores compared to those in the mild and moderate groups. The species richness of intestinal microorganisms in the severe FC group was also significantly higher, and obvious differences in the flora composition existed. Specifically, the Bacteroidota was more abundant in the severe FC group, which was a characteristic feature distinguishing severe FC. Metabolomic analyses also revealed metabolite differences among patients with mild-to-moderate and severe FC, with the severe FC group showing increased enrichment in L-isoleucine biosynthesis and glycolysis metabolic pathways. The short-chain fatty acid-targeted metabolome suggested that a decrease in butyric acid might be related to worsening constipation. This study suggests that specific flora and metabolic pathways could serve as potential diagnostic and therapeutic targets, thereby contributing to the development of new diagnostic and therapeutic approaches to improve the quality of life and therapeutic outcomes for FC patients.© 2024 The Societies and John Wiley & Sons Australia, Ltd.