Hyperglycemia enhancing the intracellular levels of reactive oxygen species (ROS) contributes to dysfunction and progressive loss of beta cells and thereby to diabetes mellitus. The oxidation sensitive calcium/calmodulin dependent phosphatase calcineurin promotes pancreatic beta cell function and survival whereas the dual leucine zipper kinase (DLK) induces apoptosis. Therefore, it was studied whether calcineurin interferes with DLK action. In a beta cell line similar concentrations of H2O2 decreased calcineurin activity and activated DLK. DLK interacted via its φLxVP motif (aa 362-365) with the interface of the calcineurin subunits A and B. Mutation of the Val prevented this protein protein interaction, hinting at a distinct φLxVP motif. Indeed, mutational analysis revealed an ordered structure of DLK's φLxVP motif whereby Val mediates the interaction with calcineurin and Leu maintains an enzymatically active conformation. Overexpression of DLK wild-type but not the DLK mutant unable to bind calcineurin diminished calcineurin-induced nuclear localisation of the nuclear factor of activated T-cells (NFAT), suggesting that both, DLK and NFAT compete for the substrate binding site of calcineurin. The calcineurin binding-deficient DLK mutant exhibited increased DLK activity measured as phosphorylation of the downstream c-Jun N-terminal kinase, inhibition of CRE-dependent gene transcription and induction of apoptosis. These findings show that calcineurin interacts with DLK; and inhibition of calcineurin increases DLK activity. Hence, this study demonstrates a novel mechanism regulating DLK action. These findings suggest that ROS through inhibition of calcineurin enhance DLK activity and thereby lead to beta cell dysfunction and loss and ultimately diabetes mellitus.