UDP-glucuronosyltransferase (UGT) 1A1-catalyzed glucuronidation is an important elimination pathway of flavonoids, and mutually inhibitory interactions may occur when two or more flavonoids are coadministered. Our recent research suggested that glucuronidation of flavonoids displayed distinct positional preferences, but whether this will lead to the mutually regioselective inhibition of UGT1A1-mediated glucuronidation of flavonoids is unknown. Therefore, we chose three monohydroxyflavone isomers, 3-hydroxyflavone (3HF), 7-hydroxyflavone (7HF), and 4'-hydroxyflavone (4'HF), and one trihydroxyflavone, 3,7,4'-trihydroxyflavone (3,7,4'THF), as the model compounds to characterize the possible mutually regioselective inhibition of glucuronidation using expressed human UGT1A1. Apparent kinetic parameters [e.g., reaction velocity (V), Michaelis-Menten constant (Km), maximum rate of metabolism (Vmax), concentration at which inhibitor achieves 50% inhibition (IC50), and the Lineweaver-Burk plots were used to evaluate the apparent kinetic mechanisms of inhibition of glucuronidation. The results showed that UGT1A1-mediated glucuronidation of three monohydroxyflavones (i.e., 3HF, 7HF, and 4'HF) and 3,7,4'THF was mutually inhibitory, and the mechanisms of inhibition appeared to be the mixed-typed inhibition. Specifically, the inhibitory effects displayed certain positional preference. Glucuronidation of 3HF was more easily inhibited by 3,7,4'THF than that of 7HF or 4'HF. Compared to 7-O-glucuronidation of 3,7,4'THF, 3-O-glucuronidation of 3,7,4'THF was more inhibited by 3HF and 4'HF, whereas glucuronidation at both 3-OH and 7-OH positions of 3,7,4'THF was more easily inhibited by 7HF than by 3HF and 4'HF. In conclusion, 3HF, 7HF, 4'HF, and 3,7,4'THF were both substrates and inhibitors of UGT1A1, and they exhibited mutually regioselective inhibition of UGT1A1-mediated glucuronidation via a mixed-type inhibitory mechanism.