Our previous studies demonstrated that the proinflammatory peptide macrophage migration inhibitory factor (MIF) functions as an autocrine mediator of both growth factor and integrin-dependent sustained ERK MAP kinase activation, cyclin D1 expression, and cell cycle progression. We now report that MIF promotes the activation of the canonical MAP kinase cascade and cyclin D1 expression by stimulating Rho guanosine triphosphatase (GTPase) activity and actin stress fiber formation. Rho-dependent stress fiber accumulation promotes the sustained activation of MAP kinase and subsequent cyclin D1 expression during G1-S phase cell cycle progression. This pathway is reported to be dependent upon myosin light chain (MLC) phosphorylation, integrin clustering, and subsequent activation of focal adhesion kinase leading to sustained MAP kinase activity. Our current studies suggests that recombinant MIF induces cyclin D1 transcription/translation in a Rho, Rho kinase, MLC kinase, and MAP kinase-dependent manner in asynchronous NIH 3T3 fibroblasts. Moreover, MIF-/- murine embryonic fibroblasts display aberrant cyclin D1 expression that is linked to defective Rho activity and MLC phosphorylation. These results strongly suggest that MIF is an integral autocrine mediator of Rho GTPase-dependent signaling events and may provide mechanistic insight into how MIF regulates proliferative, migratory, and oncogenic processes.