Macrophage immigration inhibitory factor (MIF) is a ubiquitously expressed proinflammatory mediator that has also been implicated in cell growth, cell cycle and carcinogenesis. In this study, we demonstrate siRNA-mediated knockdown of MIF results in G(0)/G(1) cell cycle arrest in HEK293 cells. To elucidate the molecular mechanism of cell cycle perturbation following MIF knockdown, we employed microarray to investigate the genome-wide expression profile in MIF-deficient cells and normal cells. Quantitative real-time PCR were used to confirm the differential expression patterns of approximately 50 transcripts involved in cell cycle and signaling identified by microarray. The dynamic model of MIF-dependent signaling pathways linked to cell cycle machinery were constructed through analyzing gene expression data in the context of known biological pathways. The results demonstrate that knockdown of MIF could inhibit G(1)/S transition through inhibition of MAPK, PI3K/Akt, NFkappaB, c-Myc-dependent pathway and activation of TGFbeta, p53-dependent pathway. Meanwhile, inhibition of E2F-, NFkappaB, c-Myc and Ap-1-mediated transcription and stimulation of p53 and FoxO4 transcriptional activity will lead to differential expression of cell cycle regulators and subsequent cell cycle arrest in G(0)/G(1) phase in MIF-knockdown cells. This study provides novel insights into the pleiotropic activities of endogenous MIF, especially its essential and crucial role in cell proliferation and the cell cycle.