Diosmetin (3′, 5, 7-trihydroxy-4′-methoxyflavone), a natural flavonoid from traditional Chinese herbs, has been used in various medicinal products because of its anticancer, antimicrobial, antioxidant, estrogenic and anti-inflammatory activity. However, flavonoids could affect the metabolic enzymes and cause drug-drug interactions (DDI), reducing the efficacy of co-administered drugs and potentially resulting in serious adverse reactions. To evaluate its potential to interact with co-administered drugs, the IC50 value of phase I cytochrome P450 enzymes (CYPs), phase II UDP-glucuronyltransferases (UGTs) and hepatic uptake transporters (organic cation transporters (OCTs), organic anion transporter polypeptides (OATPs) and Na+-taurocholate cotransporting polypeptides (NTCPs)) were examined in vitro by LC-MS/MS. Diosmetin showed strong inhibition of CYP1A2 in a concentration-dependent manner. The intensity of the inhibitory effect was followed by CYP2C8, CYP2C9, CYP2C19 and CYP2E1. For CYP2A6, CYP2B6, CYP2D6 and CYP3A4, diosmetin was found to have no significant inhibitory effects, and the induction effect on CYPs was not significant. For UGTs, diosmetin had a minimal inhibitory effect. In addition, the inhibitory effects of diosmetin on OATP and OCT1 were weak, and it had little effect on NTCP. This finding indicated that drug-drug interactions induced by diosmetin may occur through co-administration of drugs metabolized by CYP1A2.