The lifetime prevalence (∼16%) and the economic burden ($100 billion annually) associated with major depressive disorder (MDD) make it one of the most common and debilitating neurobiological illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology of MDD have not been identified. Here we use whole-genome expression profiling of postmortem tissue and show significantly increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1, encoded by DUSP1, but hereafter called MKP-1) in the hippocampal subfields of subjects with MDD compared to matched controls. MKP-1, also known as dual-specificity phosphatase-1 (DUSP1), is a member of a family of proteins that dephosphorylate both threonine and tyrosine residues and thereby serves as a key negative regulator of the MAPK cascade, a major signaling pathway involved in neuronal plasticity, function and survival. We tested the role of altered MKP-1 expression in rat and mouse models of depression and found that increased hippocampal MKP-1 expression, as a result of stress or viral-mediated gene transfer, causes depressive behaviors. Conversely, chronic antidepressant treatment normalizes stress-induced MKP-1 expression and behavior, and mice lacking MKP-1 are resilient to stress. These postmortem and preclinical studies identify MKP-1 as a key factor in MDD pathophysiology and as a new target for therapeutic interventions.