Expression of the adipocyte-derived protein resistin, which is thought to play a key role in the development of insulin resistance in vivo, is regulated by a variety of hormones and mediators, including insulin and TNFα. Here we describe our use of adenovirus-mediated gene transfer to determine which transcription factors and signaling pathways affect resistin expression in 3T3-L1 adipocytes. We found that resistin expression was enhanced by overexpression of C/EBPα and suppressed by C/EBPζ, a negative regulator of C/EBPα. Additionally, C/EBPα induced resistin even in L6 myocytes. Overexpression of PPARγ markedly reduced resistin expression, whereas PPARα had no significant effect. Resistin expression was markedly suppressed by overexpression of the PI3-kinase p110α catalytic subunit and by Akt. Finally, overexpression of MEK1, MKK6, or MKK7 suppressed resistin expression. These findings indicate that resistin expression is regulated by C/EBPα and PPARγ, partly via modulation of signal transduction in the PI3-kinase and MAP kinase pathways.