Thylakoid-rich spinach extract is being used as dietary weight-loss supplements in Japan. A recent rat study has suggested that intake of thylakoid-rich spinach extract with dietary oil inhibits dietary fat absorption via binding to bile acids, which promotes excretion of bile acids in feces. While, we confirmed that a serving size of thylakoid-rich spinach extract contains a large amount of calcium (130 mg/5 g). Therefore, using rats, we evaluated whether one-time ingestion of thylakoid-rich spinach extract affects the gastrointestinal absorption of water-insoluble drugs, such as griseofulvin (GF) and indomethacin (IM), or ciprofloxacin (CPFX) that chelate with polyvalent metal cations. Pretreatment of the rats with thylakoid-rich spinach extract (100 or 300 mg/kg) for 15 min prior to oral administration of GF (50 mg/kg) or IM (10 mg/kg) did not significantly alter the pharmacokinetic properties of either drug. Meanwhile, co-administration of thylakoid-rich spinach extract (500 mg/kg) and CPFX (20 mg/kg) significantly reduced the peak plasma concentration and the area under the plasma concentration-time curve of CPFX to 25 and 40%, respectively in rats. In vitro studies demonstrated that when a mixture of thylakoid-rich spinach extract and CPFX was centrifuged, there was a significant reduction in the supernatant concentration of CPFX relative to the control. When the experiment was repeated in the presence of ethylenediaminetetraacetic acid, the concentration of CPFX was unchanged. These results suggest that the intake of thylakoid-rich spinach extract may reduce the absorption of drugs that form a chelate with polyvalent metal cations, such as CPFX.