Tumor necrosis factor-alpha (TNF-alpha) is a potent inducer of insulin resistance, and increased TNF-alpha expression is associated with impaired glucose disposal. Although insulin is the primary regulator of glucose transport in adipose, endothelin-1, a vasoconstrictor peptide that signals through the heterotrimeric G proteins Galphaq/11, potently stimulates glucose uptake in 3T3-L1 adipocytes by a mechanism independent of phosphatidylinositol (PI) 3-kinase. Here, we report that exposure of 3T3-L1 adipocytes to TNF-alpha for 48 h dose-dependently decreased endothelin-1-stimulated glucose uptake and translocation of GLUT4 to the plasma membrane. TNF-alpha exposure had no effect on endothelin-1 receptor number at the cell surface. In contrast, TNF-alpha treatment reduced the quantity of Galphaq/11 and proline-rich tyrosine kinase 2 (PYK2) and decreased endothelin-1-stimulated PYK2-Tyr402 tyrosine phosphorylation. Taken together, these results suggest that TNF-alpha-induced desensitization of endothelin-1-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes is due, at least in part, to a decreased expression of Galphaq/11, leading to a suppression in tyrosine phosphorylation of PYK2.