Background: Per- and polyfluoroalkyl substances (PFAS) are heat and stain resisting chemicals. They are persistent, bioaccumulating and spread ubiquitously. Many hotspots where humans are exposed to high levels of PFAS have been reported. A few small observational studies in humans suggest that treatment with an Anion Exchange Resin (AER) decreases serum PFAS. This first clinical controlled crossover study aimed to assess whether AER decreases perfluorooctanesulfonic acid (PFOS) in highly exposed adults. Methods: An open label 1:1 randomized treatment sequence crossover study with allocation to oral AER (cholestyramine 4 g three times daily) or observation for 12 weeks was conducted among citizens from a PFAS hotspot. Main inclusion criteria was serum PFOS > 21 ng/mL. Primary endpoint was change in serum PFOS levels between treatment and observational period. Results: In total, 45 participants were included with a mean age of 50 years (SD 13). Serum PFOS baseline median was 191 ng/mL (IQR: 129–229) and decreased with a mean of 115 ng/mL (95 % CI: 89–140) on treatment, and 4.3 ng/mL in observation period corresponding to a decrease of 60 % (95 % CI: 53–67; p < 0.0001). PFHxS, PFOA, PFNA and PFDA decreased during treatment between 15 and 44 %. No serious adverse events were reported. Conclusions: Oral treatment with AER significantly lowered serum PFOS concentrations suggesting a possible treatment for enhancing elimination of PFOS in highly exposed adults.