14-3-3 family members are dimeric phosphoserine-binding proteins that participate in signal transduction and checkpoint control pathways. In this work, dominant-negative mutant forms of 14-3-3 were used to disrupt 14-3-3 function in cultured cells and in transgenic animals. Transfection of cultured fibroblasts with the R56A and R60A double mutant form of 14-3-3ζ (DN-14-3-3ζ) inhibited serum-stimulated ERK MAPK activation, but increased the basal activation of JNK1 and p38 MAPK. Fibroblasts transfected with DN-14-3-3ζ exhibited markedly increased apoptosis in response to UVC irradiation that was blocked by pre-treatment with a p38 MAPK inhibitor, SB202190. Targeted expression of DN-14-3-3η to murine postnatal cardiac tissue increased the basal activation of JNK1 and p38 MAPK, and affected the ability of mice to compensate for pressure overload, which resulted in increased mortality, dilated cardiomyopathy and massive cardiomyocyte apoptosis. These results demonstrate that a primary function of mammalian 14-3-3 proteins is to inhibit apoptosis.