We examined whether flavonoids act as endogenous auxin transport regulators during gravity vector and light intensity changes in Arabidopsis thaliana roots. Flavonoid deficient transparent testa4 [tt4(2YY6)] seedlings had elevated root basipetal auxin transport compared with the wild type, consistent with the absence of a negative auxin transport regulator. The tt4(2YY6) roots had delayed gravitropism that was chemically complemented with a flavonoid intermediate. Flavonoid accumulation was found in wild-type columella cells, the site of gravity perception, and in epidermal and cortical cells, the site of differential growth, but flavonoid accumulation was absent in tt4(2YY6) roots. Flavonoid accumulation was higher in gravity-stimulated root tips as compared with vertical controls, with maximum differences coinciding with the timing of gravitropic bending, and was located in epidermal cells. Exogenous indole-3-acetic acid (IAA) also elevated flavonoid accumulation, suggesting that flavonoid changes in response to gravity might be partly as a result of changing IAA distribution. Acropetal IAA transport was also elevated in roots of tt4(2YY6). Flavonoid synthesis was repressed in the dark, as were differences in root acropetal transport in tt4(2YY6). These results are consistent with light- and gravity-induced flavonoid stimulation that alters auxin transport in roots and dependent physiological processes, including gravitropic bending and root development.