Macrophage migration inhibitory factor (MIF) is a well-described proinflammatory mediator. MIF overexpression has been observed in many tumors and is implicated in oncogenic transformation and tumor progression. However, the molecular mechanisms responsible for regulating MIF expression remain poorly understood. In this study, we showed that the transcriptional repressor HBP1 (HMG box-containing protein 1) negatively regulates MIF expression. We first identified a large high-affinity HBP1 DNA-binding element at positions -811 to -792 from the transcriptional start site within the MIF promoter by computer analysis. Reporter analyses showed that this element was required for HBP1-mediated transcriptional repression. Furthermore, HBP1 associated with the MIF promoter in vivo and repressed endogenous MIF gene expression. Consistent with HBP1-mediated repression of MIF, low levels of HBP1 expression were associated with high levels of MIF expression in prostate cancer samples. Importantly, HBP1-mediated repression of MIF inhibited tumorigenic growth and invasion, and the repressive effect of HBP1 on tumorigenic growth and invasion could be partially rescued by the addition of recombinant MIF to the culture medium. Finally, prostate tumor samples with low HBP1 and high MIF expression were associated with a significant decrease in relapse-free survival. Taken together, these results indicated that HBP1 directly inhibited MIF gene transcription, and suggested that the loss of HBP1 expression or activity may contribute to the upregulation of MIF expression in prostate tumor tissue.