14-3-3 family members are intracellular dimeric phosphoserine-binding proteins that regulate signal transduction, cell cycle, apoptotic, and metabolic cascades. Previous work with global 14-3-3 protein inhibitors suggested that these proteins play a critical role in antagonizing apoptotic cell death in response to provocative stimuli. To determine the specific role of one family member in apoptosis, mice were generated with targeted disruption of the 14-3-3τ gene. 14-3-3τ-/- mice did not survive embryonic development, but haploinsufficient mice appeared normal at birth and were fertile. Cultured adult cardiomyocytes derived from 14-3-3τ+/- mice were sensitized to apoptosis in response to hydrogen peroxide or UV irradiation. 14-3-3τ+/- mice were intolerant of experimental myocardial infarction and developed pathological ventricular remodeling with increased cardiomyocyte apoptosis. ASK1, c-jun NH2-terminal kinase, and p38 mitogen-activated protein kinase (MAPK) activation was increased, but extracellular signal-regulated kinase MAPK activation was reduced, in 14-3-3τ+/- cardiac tissue. Inhibition of p38 MAPK increased survival in 14-3-3τ+/- mice subjected to myocardial infarction. These results demonstrate that 14-3-3τ plays a critical antiapoptotic function in cardiomyocytes and that therapeutic agents that increase 14-3-3τ activity may be beneficial to patients with myocardial infarction.