Constipation, a widespread gastrointestinal disorder, often leads to the exploration of natural remedies. This study examines the efficacy of Golden Flower Tibetan Tea Polysaccharides (GFTTPs) in alleviating constipation in mice. Chemical analyses reveal that GFTTPs possess O-H, carboxyl, carboxylic acid (-COOH), and C-O-C groups, alongside a porous crystal structure with thermal stability. In animal experiments, GFTTPs significantly upregulated aquaporin 3 (AQP3) and aquaporin 8 (AQP8) expressions in the colon, enhancing water absorption and reducing fecal water content. At a 400 mg/kg dosage, GFTTPs notably improved colonic tissue alterations and serum levels of excitatory neurotransmitters caused by loperamide hydrochloride. They also beneficially altered gut microbiota, increasing Coprococcus, Lactobacillus, and Pediococcus populations. These changes correlated with improved stool frequency, consistency, and weight in constipated mice. Importantly, GFTTPs at 200 and 400 mg/kg doses exhibited comparable effects to the normal control group in key parameters, such as gastrointestinal transit rate and fecal moisture. These findings suggest that GFTTPs may serve as a potent natural remedy for constipation, offering significant therapeutic potential within the context of gut health and with promising implications for human applications.