The macrophage migration inhibitory factor (MIF) is a hypoxia regulated gene that has a variety of tumorigenic functions. In clear cell renal carcinoma (CCRC), hypoxic signaling is constitutively active because of the frequent loss of function of the von Hippel-Lindau tumor suppressor protein. We therefore sought to assess the expression of MIF in CCRC and its biological functions. We stained tumor tissue microarrays comprising sections of 128 CCRC tumors and found MIF to be moderately or highly expressed in >98%. MIF expression was further found to be dramatically elevated in blood plasma of individuals with CCRC compared with healthy controls, suggesting that measurement of MIF levels in the blood may have utility as a diagnostic marker in CCRC. At a functional level, MIF has been reported to engage the CD74 and CD44 receptors and induce signal transduction. In CCRC cell lines, depletion of MIF, CD74 or CD44 by small hairpin RNA led to a significant reduction in growth rate, and clonogenic survival, coinciding with the degree of knockdown. Interruption of the MIF pathway also decreased tumorigenic potential. Biochemically, we found that in CCRC cells MIF signaling leads to activation of the mitogen-activated protein kinase pathway and to Src phosphorylation, which is critical for regulation of p27. Together, our studies establish MIF as a protumorigenic signaling molecule that functions in an autocrine fashion to promote renal cell carcinoma and may be useful as a minimally invasive marker of disease status.