TWEAK (TNF-like weak inducer of apoptosis), a relatively new member of the TNF superfamily, is an important immune/inflammatory regulator that has different functional properties to that of other members of this superfamily. We report herein that TWEAK induces cellular insulin resistance in both human hepatocellular carcinoma cell lines (Huh7 and HepG2) and primary rat hepatocytes by inhibiting both early insulin receptor (IR) signaling events and the downstream actions of insulin. TWEAK profoundly inhibited insulin-induced Akt phosphorylation in both a concentration- and time-dependent manner. This inhibitory effect occurred via mechanisms that involved the TWEAK receptor Fn14 and the activation of the canonical and non-canonical NF-B signaling pathways. Furthermore, TWEAK significantly inhibited IR autophosphorylation and IRS-1 activation, with concomitant increases in serine phosphorylation of IRS-1. Moreover, insulin-induced reduction of gluconeogenic enzyme gene expression and increases in glycogen synthesis in hepatocytes were significantly attenuated by TWEAK treatment. Therefore, these findings not only reveal a novel pathophysiological function of TWEAK/Fn14, but also uncover a new player that may contribute to the development of cellular insulin resistance in hepatocytes.