Microsomal triglyceride transfer protein (MTP) is rate limiting for the assembly and secretion of apolipoprotein B-containing lipoproteins. Elevated hepatic MTP mRNA level, presumably as a result of impaired insulin signaling, has been implicated in the pathophysiology of dyslipidemia associated with insulin resistance/type 2 diabetes. In this study, we showed that insulin decreases MTP mRNA level mainly through transcriptional regulation in HepG2 cells. We further characterized the corresponding signal transduction pathway, using chemical inhibitors and constitutively active and dominant negative forms of regulatory enzymes. We demonstrated that insulin inhibits MTP gene transcription through MAPKerk cascade but not through the PI 3-kinase pathway. Activation of ras through farnesylation is not a prerequisite for the inhibition. In addition, cellular MAPKerk and MAPKp38 activities play a counterbalancing role in regulating the MTP gene transcription. These complex regulations may represent a means to fine-tuning MTP gene transcription in response to a diverse set of environmental stimuli and may have important implications for the onset and development of diabetes-associated dyslipidemia.