ABSTRACTPlants have evolved a number of adaptive strategies to cope with fluctuations in phosphorus (P) supply. The current knowledge of the transcriptional regulation of the P‐starvation response in plants is limited. However, one MYB‐related transcription factor, PHR1, is known to be involved in the P‐starvation response. In this paper, we characterize a T‐tagged phr1 knockout mutant and a series of transgenic plant lines which over‐express PHR1 in wild type (WT) and phr1 mutant background. The knockout mutant has an altered phosphate (Pi) allocation between root and shoot; accumulates less anthocyanins, sugars and starch than P‐starved WT; has a lower AGPase activity; and is impaired in induction of a subset of Pi starvation‐induced genes. Expression of PHR1 in the phr1 mutant rescues the responsiveness to P‐starvation and leads to WT levels of sugars and starch during Pi starvation conditions, confirming the involvement of PHR1 in adjusting carbon metabolism. Over‐expression of PHR1 further resulted in a dramatic increase in the microRNA miR399d, and this resulted in changes in the transcript level for the target gene PHO2. Furthermore, over‐expression of PHR1 in both WT and phr1 mutant results in strongly increased content of Pi irrespective of P regime. This shows that targeting a key regulatory element in the Pi starvation regulatory network represents a useful approach for molecular breeding of plants towards more efficient Pi uptake and assimilation.