We have shown previously that macrophage migration inhibitory factor (MIF) may play a role in the destabilization of atherosclerotic plaques by activating matrix metalloproteinase protein-9 (MMP-9). The aim of this study is to investigate the signaling mechanism by which MIF induces MMP-9 expression and activation in a murine macrophage line (RAW264.7). MIF was able to activate extracellular signal-regulated kinase 1/2 (ERK1/2), to a less extent JNK, but not p38 mitogen-activated protein (MAP), MAP kinase to induce MMP9 mRNA and protein expression in RAW264.7 murine macrophages. This was confirmed by the findings that addition of an ERK MAP kinase inhibitor (PD98059) but not a p38 inhibitor (SB203589) abolished MIF-induced MMP-9 expression and activation, whereas addition of a JNK inhibitor (SP600125) produced a partially inhibitory effect. The functional role of mitogen-activated protein kinase kinase (MEK)-ERK MAP kinase in MIF-induced MMP-9 expression was further confirmed by overexpressing dominant negative MEK (DN-MEK) and DN-ERK MAP kinases. Interestingly, constitutive expression of a wild-type (WT)-MEK alone was also capable of inducing a low, but significant MMP-9 mRNA and protein expression but did not cause a further increase in MMP-9 in response to MIF. MIF activates the MEK-ERK MAP kinase pathway to induce MMP-9 expression by murine macrophages. Activation of this pathway is necessary for MMP-9 expression and activation in response to MIF stimulation.