AbstractVegetables and fruits contain prenylflavonoids with biological functions that might improve human health. The prenylflavonoid isoxanthohumol (IXA) and its derivative, 8‐prenylnaringenin (8‐PN), have beneficial activities, including anti‐cancer effects and suppression of insulin resistance. However, their pharmacokinetic profile is unclear. Previous studies suggested flavonoids have low systemic availability and are excreted via the feces. Therefore, this study investigated the tissue distribution dynamics of high‐purity IXA (>90%) from hops administered orally, either singly (50 mg/kg body weight [BW]) or daily for 14 days (30 mg/kg BW), to mice. High‐pressure liquid chromatography demonstrated that IXA was absorbed rapidly after a single administration and reached plasma maximum concentration (Cmax) (3.95 ± 0.81 μmol/L) by 0.5 h. IXA was present at high levels in the liver compared with the kidney, pancreas, lung, skeletal muscle, spleen, thymus, and heart. The highest IXA level after 14 days of IXA ingestion was observed in the liver, followed by the kidney, thymus, spleen, lung, and brain. There was no significant difference in IXA accumulation in tissues between the single and multiple dose groups. Analyses of the livers of rats treated with different concentrations of IXA (112.5–1500 mg/kg BW) once a day for 28 days demonstrated that IXA accumulated dose‐dependently with a correlation coefficient of .813. The accumulation of 8‐PN was dependent on the intake period but not the intake amount of IXA (correlation coefficient −.255). In summary, IXA and 8‐PN were detected in tissues and organs up to 24 h after ingestion, suggesting that orally ingested IXA might have health benefits as a nutraceutical.