Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells. We have shown that ET-1 stimulates the adaptor protein p66Shc through Rac/Cdc42 guanine nucleotide exchange factor β₁Pix. In this study, we demonstrate that ET-1-induced serine phosphorylation of p66Shc is mediated through Gα_i3. Pertussis toxin treatment of cells induced a significant decrease in the interaction between β₁Pix and ET_A-R, and an increase in the binding of Gα_i3 and G_β1 to β₁Pix. Activation of heterotrimeric G proteins by AlF₄⁻ resulted in an increase of Gα_i3 binding to β₁Pix, which was significantly disrupted in cells expressing β₁Pix dimerization deficient mutant, β₁PixΔ (602-611). In cells expressing β₁PixΔ (602-611), ET-1-induced p66Shc activation was also significantly decreased. Specific inhibition of EGF receptor by AG1478 blocked ET-1-induced p66Shc activation and the binding of p66Shc and Gα_i3 to β₁Pix. Inhibition of Erk1/2 blocked p66Shc activation induced by ET-1. Altogether, our results indicate that ET-1 activates p66Shc through EGF receptor transactivation, leading to the activation of Gα_i3, β₁Pix and Erk1/2.