Macrophage migration inhibitory factor (MIF) is a potent modulator of the p53 signaling pathway, but the molecular mechanisms of the effect of MIF on p53 function have so far remained unclear. Here we show that MIF physically interacts with the p53 tumor suppressor in vitro and in vivo. This association was significantly reduced by a C81S mutation but not C57S or C60S mutations, suggesting that Cys(81) is essential for the in vivo association between MIF and p53. This association also depended on Cys(242) (and, to some extent, on Cys(238)) within the central DNA binding domain of p53. Ectopic expression of MIF, but not MIF(C81S), inhibited p53-mediated transcriptional activation in a dose-dependent manner. Conversely, knockdown of endogenous MIF stimulated p53-mediated transcription. MIF inhibited p53-induced apoptosis and cell cycle arrest, whereas the MIF(C81S) mutant, which is unable to physically associate with p53, had no effect. Consistent with these findings, confocal microscopy showed that MIF prevented p53 translocation from the cytoplasm to the nucleus. We also demonstrated that MIF suppresses p53 activity by stabilizing the physical association between p53 and Mdm2. These results suggest that MIF physically associates with p53 and negatively regulates p53 function.