The response of plants to Pi limitation involves interplay between root uptake of Pi, adjustment of resource allocation to different plant organs and increased metabolic Pi use efficiency. To identify potentially novel, early-responding, metabolic hallmarks of Pi limitation in crop plants, we studied the metabolic response of barley leaves over the first 7 d of Pi stress, and the relationship of primary metabolites with leaf Pi levels and leaf biomass. The abundance of leaf Pi, Tyr and shikimate were significantly different between low Pi and control plants 1 h after transfer of the plants to low Pi. Combining these data with 15N metabolic labelling, we show that over the first 48 h of Pi limitation, metabolic flux through the N assimilation and aromatic amino acid pathways is increased. We propose that together with a shift in amino acid metabolism in the chloroplast a transient restoration of the energetic and redox state of the leaf is achieved. Correlation analysis of metabolite abundances revealed a central role for major amino acids in Pi stress, appearing to modulate partitioning of soluble sugars between amino acid and carboxylate synthesis, thereby limiting leaf biomass accumulation when external Pi is low.