Obesity is a condition in which adipose tissue mass is expanded. Increases in both adipocyte size and number contribute to enlargement of adipose tissue. The increase in cell number is thought to be caused by proliferation and differentiation of preadipocytes. Macrophage migration inhibitory factor (MIF) is expressed in adipocytes, and intracellular MIF content is increased during adipogenesis. Therefore, we hypothesized that MIF is associated with adipocyte biology during adipogenesis and focused on the influence of MIF on adipogenesis. To examine the effects of MIF on adipocytes, MIF expression in 3T3-L1 preadipocytes was inhibited by RNA interference, and cell differentiation was induced by standard procedures. The triglyceride content of MIF small interfering RNA (siRNA)-transfected 3T3-L1 cells was smaller than that of nonspecific siRNA-transfected cells. In addition, MIF knockdown apparently abrogated increases in adiponectin mRNA levels during differentiation. Gene expression of peroxisome proliferator-activated receptor (PPAR)gamma, CCAAT/enhancer binding protein (C/EBP)alpha, and C/EBPdelta decreased with MIF siRNA transfection, but C/EBPbeta expression increased. Cell number and incorporation of 5-bromo-2-deoxyuridine into cells decreased from 1-3 d and from 14-20 h, respectively, after induction of differentiation in MIF siRNA-transfected cells, thus suggesting that MIF siRNA inhibits mitotic clonal expansion. Taken together, these results indicated that MIF regulates differentiation of 3T3-L1 preadipocytes, at least partially, through inhibition of mitotic clonal expansion and/or C/EBPdelta expression.