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The PK and tissue distribution of 5,7-DMF was evaluated for the first time in micePeak 5,7-DMF concentrations in mouse plasma and tissues were reached within 30 min.5,7-DMF oral dose led to extensive tissue distribution.5,7-DMF was abundant in gut > liver > kidney > brain > spleen > heart > lung > adipose > muscle.The partition coefficient (Kp) of these tissues were 0.65 to 12.9.5,7-Dimethoxyflavone (5,7-DMF) is a major active constituent of many herbal plants, such as Kaempferia paviflora, Piper caninum, and Leptospermum scoparium. 5,7-DMF has demonstrated many beneficial pharmacological effects in vitro, including anti-infammatory, anti-oxidant, cardioprotection effects, as well as chemopreventive and chemosensitizing properties. In contrast to the extensive in vitro investigations, the information of the pharmacokinetic (PK) profile of 5,7-DMF in vivo is very limited. In this study we investigated the PK and tissue distribution of 5,7-DMF in mice following single oral dose of 10 mg/kg 5,7-DMF. Mouse plasma, heart, lung, liver, kidney, intestine, brain, spleen, muscle and fat tissues were collected and analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Maximal 5,7-DMF concentrations in plasma and tissues were reached within 30 min. The peak plasma concentration (Cmax) was 1870 ± 1190 ng/mL, and area under the curve (AUCt) was 532 ± 165 h ng/mL and terminal half-life was 3.40 ± 2.80 h. The volume of distribution was 90.1 ± 62.0 L/kg. Clearance was 20.2 ± 7.5 L/h/kg. Except for muscle and adipose, other tissues had higher Cmax than plasma, ranging from 1.75- to 9.96-fold. After oral administration, 5,7-DMF was most abundant in gut, followed by liver, kidney, brain, spleen, heart, lung, adipose and muscle. The partition coefficient (Kp) of these tissues were 0.65 to 12.9. In conclusion, we reported for the first time the PK and tissue distribution of 5,7-DMF in mice. These results will be critical in evaluating if those beneficial in vitro effects can be translated in vivo.