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Abstract Diketopiperazines (DKPs) are chemically and functionally diverse cyclic dipeptides associated primarily with microbes. Few DKPs have been reported from plants and animals; the best characterized is cyclo(His-Pro), found in the mammalian central nervous system, where it arises from the proteolytic cleavage of a thyrotropin-releasing tripeptide hormone. Herein, we report the identification of cyclo(His-Pro) in Arabidopsis (Arabidopsis thaliana), where its levels increase upon abiotic stress conditions, including high salt, heat, and cold. To screen for potential protein targets, we used isothermal shift assays, which examine changes in protein-melting stability upon ligand binding. Among the identified proteins, we focused on the glycolytic enzyme, cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC1). Binding between the GAPC1 protein and cyclo(His-Pro) was validated using nano-differential scanning fluorimetry and microscale thermophoresis, and we could further demonstrate that cyclo(His-Pro) inhibits GAPC1 activity with an IC50 of ∼200 μm. This inhibition was conserved in human GAPDH. Inhibition of glyceraldehyde-3-phosphate dehydrogenase activity has previously been reported to reroute carbon from glycolysis toward the pentose phosphate pathway. Accordingly, cyclo(His-Pro) supplementation augmented NADPH levels, increasing the NADPH/NADP+ ratio. Phenotypic screening revealed that plants supplemented with cyclo(His-Pro) were more tolerant to high-salt stress, as manifested by higher biomass, which we show is dependent on GAPC1/2. Our work reports the identification and functional characterization of cyclo(His-Pro) as a modulator of glyceraldehyde-3-phosphate dehydrogenase in plants.