Recent studies have implicated the endogenous opioid system in the antidepressant actions of ketamine, but the underlying mechanisms remain unclear. We used a combination of pharmacological, behavioral, and molecular approaches in rats to test the contribution of the prefrontal endogenous opioid system to the antidepressant-like effects of a single dose of ketamine. Both the behavioral actions of ketamine and their molecular correlates in the medial prefrontal cortex (mPFC) were blocked by acute systemic administration of naltrexone, a competitive opioid receptor antagonist. Naltrexone delivered directly into the mPFC similarly disrupted the behavioral effects of ketamine. Ketamine treatment rapidly increased levels of β-endorphin and the expression of the μ-opioid receptor gene (Oprm1) in the mPFC, and the expression of the gene that encodes proopiomelanocortin, the precursor of β-endorphin, in the hypothalamus, in vivo. Finally, neutralization of β-endorphin in the mPFC using a specific antibody prior to ketamine treatment abolished both behavioral and molecular effects. Together, these findings indicate that presence of β-endorphin and activation of opioid receptors in the mPFC are required for the antidepressant-like actions of ketamine.