Exposure to per- and polyfluoroalkyl substances (PFASs) is a major concern due to their widespread occurrence and adverse health outcomes. The possible binding of PFASs to peroxisome proliferator-activated receptors (PPARs) and nuclear receptors raises concerns that PFASs may impact cholesterol levels in human. In this study, the U.S. National Health and Nutrition Examination Survey (NHANES) data were employed to address the temporal trend for PFAS concentrations in biomonitoring and associations between cholesterol levels and PFAS exposure. Compared to the PFAS levels in 2003–2004, the median perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS) and perfluorononanoic acid (PFNA) levels in human serum in 2013–2014 decreased from 3.7 to 1.8 ng/mL, 19.2–4.7 ng/mL, 1.7 ng/mL to 1.3 ng/mL and 0.8 ng/mL to 0.6 ng/mL, respectively. Also, an estimate of 1.5 ± 0.7 mg/dL (95% confidence interval: 0.2 − 2.8) and 0.4 ± 0.2 mg/dL (95% confidence interval: 0.1 – 0.6) total cholesterol increases for unit PFOA and PFOS increase (ng/mL), respectively. By using hybrid approach, RfDs were estimated to be 2.0 ng PFOS/kg per day and 0.8 ng PFOA/kg per day. However, it should be cautious when using proposed RfDs based on data obtained from cross-sectional datasets, especially evidence based on data originating from experimental or animal studies is still controversial.