Phosphate (Pi) deficiency significantly limits plant growth in natural and agricultural systems. Accumulation of anthocyanins in shoots is a common response of Arabidopsis thaliana to Pi deficiency. To elucidate the mechanisms underlying Pi deficiency-induced anthocyanin accumulation, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ) to investigate protein expression profiles of Arabidopsis thaliana seedlings subjected to Pi deficiency for 7 days. In total, 5,106 proteins were identified, of which 156 displayed significant changes in abundance upon Pi deficiency. Bioinformatics analysis indicated that flavonoid biosynthesis was the most significantly elevated metabolic process under Pi deficiency. We further examined the potential role of the flavonoid biosynthetic pathway using a dihydroflavonol 4-reductase (DFR) mutant (tt3) and quantitative RT-PCR (qRT-PCR) analysis, and found that the tt3 mutant was deprived of transcriptional up-regulation of three genes related to anthocyanin biosynthesis, modification and transport under Pi deficiency. These results showed that Pi deficiency probably enhances the anthocyanin accumulation by promoting the flavonoid biosynthesis. The exact functions of these proteins remain to be examined. Nevertheless, our study increases the understanding of the mechanisms implicated in the anthocyanin accumulation induced by Pi deficiency and adaptive responses of plants to Pi starvation.