Although it is well known that phosphate (Pi) deficiency affects flavonoid accumulation in higher plants, knowledge on the regulation and potential function of flavonoids in the plants grown with low Pi supply is lacking. In this work, we found that low Pi treatment caused significant reduction of root flavonoid (e.g. quercetin, kaempferol and their derivatives) levels in both Columbia (Col-0) and Landsberg erecta (Ler) ecotypes of Arabidopsis thaliana (L.) Heynh. Further investigations revealed that the dysfunction of PHR1, PHO1, PHO2 and NPC4 did not affect the decrease of root flavonoid level by low Pi treatment. In contrast, pldζ2, a knockout mutant of the Arabidopsis phospholipase Dζ2, exhibited defects in the reduction of root flavonoid level and lateral root (LR) emergence under low Pi conditions. When grown under low Pi supply, the transport of auxin from the shoot apex into the root, expression of the auxin responsive DR5::GUS marker and induction of the auxin responsive genes were all significantly less efficient in pldζ2 than in wild-type (WT) control. This is the first report on the reduction of root flavonoid level and its likely contribution to increased LR emergence in Arabidopsis under Pi deficiency conditions, which may facilitate the adaptation of plants to the growth environments with poor Pi availability.