Constipation is one of the most prevalent gastrointestinal disorders worldwide. Recent studies have demonstrated that probiotics may alleviate constipation by restoring gut microbiota balance. This study investigated the effects of BC99 (formerly BC99) on gut microbiota and intestinal function in a loperamide-induced mouse model of functional constipation. BALB/c mice were randomly divided into six groups: control, model, phenolphthalein, BC99-L (2 × 10 CFU/day), BC99-M (2 × 10 CFU/day), and BC99-H (2 × 10 CFU/day). After 14 days of supplementation, constipation was induced in mice via loperamide administration. BC99 significantly increased fecal water content, gastrointestinal transit rate, microbial metabolic activity, and butyric acid production, while decreasing the abundance of inflammation-related metabolic pathways. Moreover, BC99 improved levels of gastrointestinal regulatory peptides, including motilin and somatostatin. The Firmicutes-to-Bacteroidetes ratio was elevated in the BC99-M and the BC99-H group compared to the model group, indicating that BC99 effectively modulated gut microbiota composition and host biosynthetic pathways. BC99 alleviated and prevented loperamide-induced functional constipation in mice by enhancing intestinal peristalsis and restoring gut microbial homeostasis.