Food is a major source of human exposure to perfluorooctanoic acid (PFOA), however, PFOA bioavailability in food has not been studied. An in vivo mouse model and three in vitro methods (unified BARGE method, UBM; physiologically based extraction test, PBET; and in vitro digestion method, IVD) were used to determine the relative bioavailability and bioaccessibility of PFOA in the presence of 17 foods. PFOA was mixed with foods of different nutritional compositions and fed to mice over a 7-d period. PFOA relative bioavailability was determined by comparing PFOA accumulation in the liver following PFOA exposure via food to that in water. PFOA bioavailability relative to water ranged from 4.30 ± 0.80 to 69.0 ± 11.9% and was negatively correlated with lipid content (r = 0.76). This was possibly due to competitive sorption of free fatty acids with PFOA onto transporters on intestine epithelial cells. Besides, cations in the gastrointestinal tract, such as Ca(2+) and Mg(2+), are capable of complexing PFOA and partitioning to the lipid phase. On the other hand, when assessed using in vitro assays, PFOA bioaccessibility varied with methods, being 8.7-73% (UBM), 9.8-99% (PBET), and 21-114% (IVD). PFOA bioaccessibility was negatively correlated with lipid content when assessed using UBM (r = 0.82); however, a poor correlation with food composition was observed for PBET and IVD (r = 0.01-0.50). When in vivo and in vitro data were compared, a strong correlation was observed for UBM (r = 0.79), but poor relationships were observed for PBET and IVD (r = 0.11-0.22). This was probably because the higher lipolysis ability and presence of Ca(2+) and Mg(2+) in the gastrointestinal fluid of UBM resulted in a lower potential to form stable micelles compared to PBET and IVD. These results indicated that PFOA relative bioavailability was mainly affected by lipid content in foods, and UBM has the potential to determine PFOA bioaccessibility in food samples.