Fluoride exposure primarily occurs through contaminated water and leads to fluorosis, which is a global health concern. After ingestion, fluoride is absorbed via gastrointestinal tract, where it interacts with the gut microbiota. While animal studies have explored fluoride's effects on gut microbiota, no human studies have yet been conducted. Most research emphasizes metagenomic diversity, neglecting isolation and characterization of pure cultures for further applications. Additionally, the association between gut microbiota with fluorosis outcomes in fluoride-exposed populations is unexplored. This study characterizes and compares the cultivable gut microbiota in the fluoride-exposed population with (symptomatic, group II) or without (asymptomatic, group I) signs of skeletal fluorosis along with unexposed control (group III). Group I displayed higher abundance of Firmicutes (58.58%), group II had predominance of Proteobacteria (61.25%) while group III showed similar abundance of Proteobacteria (50.38%) and Firmicutes (49.51%). On analyzing short-chain fatty acid (SCFA) profiles, group I isolates produced higher isobutyric acid (1.31 ± 0.9 mM) than group II (0.71 ± 0.35 mM), while group II produced more isovaleric acid (0.8 ± 0.41 mM) than group I (0.61 ± 0.08 mM) (p <
0.05). These findings suggest that gut microbiota and SCFAs alteration may influence bone metabolism, affecting the fluorosis progression.