Auxin determines the developmental fate of plant tissues, and local auxin concentration is precisely controlled. The role of auxin transport in modulating local auxin concentration has been widely studied but the regulation of local auxin biosynthesis is less well understood. Here, we show that TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1), a key enzyme in the auxin biosynthesis pathway in Arabidopsis thaliana is phosphorylated at Threonine 101 (T101). T101 phosphorylation status can act as an on/off switch to control TAA1-dependent auxin biosynthesis and is required for proper regulation of root meristem size and root hair development. This phosphosite is evolutionarily conserved suggesting post-translational regulation of auxin biosynthesis may be a general phenomenon. In addition, we show that auxin itself, in part via TRANS-MEMBRANE KINASE 4 (TMK4), can induce T101 phosphorylation of TAA1 suggesting a self-regulatory loop whereby local auxin signalling can suppress biosynthesis. We conclude that phosphorylation-dependent control of TAA1 enzymatic activity may contribute to regulation of auxin concentration in response to endogenous and/or external cues.