Exposure to cigarette smoke (CS) is the most common cause of emphysema, a debilitating pulmonary disease histopathologically characterized by the irreversible destruction of lung architecture. Mounting evidence links enhanced endothelial apoptosis causally to the development of emphysema. However, the molecular determinants of human endothelial cell apoptosis and survival in response to CS are not fully defined. Such determinants could represent clinically relevant targets for intervention. We show here that CS extract (CSE) triggers the death of human pulmonary macrovascular endothelial cells (HPAECs) through a caspase 9–dependent apoptotic pathway. Exposure to CSE results in the increased expression of p53 in HPAECs. Using the p53 inhibitor, pifithrin-α (PFT-α), and RNA interference (RNAi) directed at p53, we demonstrate that p53 function and expression are required for CSE-mediated apoptosis. The expression of macrophage migration inhibitory factor (MIF), an antiapoptotic cytokine produced by HPAECs, also increases in response to CSE exposure. The addition of recombinant human MIF prevents cell death from exposure to CSE. Further, the suppression of MIF or its receptor/binding partner, Jun activation domain–binding protein 1 (Jab-1), with RNAi enhances the sensitivity of human pulmonary endothelial cells to CSE via a p53-dependent (PFT-α–inhibitable) pathway. Finally, we demonstrate that MIF is a negative regulator of p53 expression in response to CSE, placing MIF upstream of p53 as an antagonist of CSE-induced apoptosis. We conclude that MIF can protect human vascular endothelium from the toxic effects of CSE via the antagonism of p53-mediated apoptosis.