Diphospho-myo-inositol polyphosphates, also termed inositol pyrophosphates, are molecular messengers containing at least one high-energy phosphoanhydride bond and regulate a wide range of cellular processes in eukaryotes. While inositol pyrophosphates InsP and InsP are present in different plant species, both the identity of enzymes responsible for InsP synthesis and the isomer identity of plant InsP remain unknown. This study demonstrates that Arabidopsis ITPK1 and ITPK2 catalyze the phosphorylation of phytic acid (InsP) to the symmetric InsP isomer 5-InsP and that InsP kinase activity of ITPK enzymes is evolutionarily conserved from humans to plants. We also show by P NMR that plant InsP is structurally identical to the in vitro InsP kinase products of ITPK1 and ITPK2. Our findings lay the biochemical and genetic basis to uncover physiological processes regulated by 5-InsP in plants.