Constipation is a globally prevalent functional digestive disorder characterized by the difficult and lack of periodicity during defecating. However, the pathophysiology of functional constipation is attributed to multiple factors and the associated diagnosis and treatments are still poorly understood. Colon hydrotherapy (CHT) has emerged as an alternative therapy for constipation in recent years, which promotes intestinal peristalsis and improves stool consistency. Here, we retrospectively analyzed the clinical and pathological data of 99 enrolled constipation patients who were treated with conventional drugs or CHT. Results showed that CHT rendered the similar therapeutic efficacy as conventional laxatives in the treatment of conventional constipation. CHT also showed better relief of constipation symptoms among patients with refractory constipation than patients with the conventional constipation. Moreover, CHT significantly reduced the constipation score and improved the quality of life in refractory constipation patients who were resistant to conventional laxatives. Through 16S rDNA sequencing and fecal microbiota transplantation (FMT), we demonstrated that CHT effectively altered the overall composition and community structure of gut microbiota among patients. FMT from patients with refractory constipation significantly aggravated the symptoms of loperamide-induced constipation mice, including physiological and biochemical parameters. Furthermore, we confirmed that CHT greatly relieve anxiety and depressive behavior associated with constipation by using the self-designed mouse colon hydrotherapy device. Our results suggest that CHT could be a promising therapeutic strategy for constipation patients, particularly for those who have failed in conventional drug treatments.