Antimony (Sb) exposure threatens human health. To identify human biomarkers for Sb exposure, we analyzed 480 environmental samples from an active Sb mining area in Hunan, China. Elevated Sb concentrations exceeding the reference level were detected in drinking water (70% of n = 83 total samples), foods (80%, n = 188), urine (95%, n = 63), saliva (44%, n = 48), hair (80%, n = 51) and nails (83%, n = 47). Drinking water contributed 85%–100% of the average daily dose (ADD) of Sb, and the total ADD (11.7 μg/kg bodyweight/day) was up to thirty times higher than the oral reference dose (0.4 μg/kg bodyweight/day) as recommended by USEPA. A positive correlation was found between ADD and Sb content in hair (p = 0.02), but not in urine (p = 0.051), saliva (p = 0.52) or nails (p = 0.85), suggesting that hair is the best non-invasive biomarker. Micro X-ray fluorescence analysis indicated that Sb is distributed in discrete spots in hair and nails, and Sb distribution is correlated with other metals. Methylated Sb species were predominant in urine (46%–100%) and saliva (74%–100%) in collected samples, implying that the human metabolic system adopts methylation as an effective pathway to detoxify and excrete Sb. Display omitted •Sb content in water and food near the mining area is higher than the reference level.•Hair can be used as a non-invasive biomarker for Sb exposure.•Interaction effects explained most of the Sb concentration in biomarkers.•Methylated Sb was the predominant species in urine and saliva.