The phosphorylation of forkhead transcription factor FOXO3a by Akt is critical regulator of cell proliferation induced by serum. We show that endothelin-1 (ET-1) stimulation of primary human mesangial cells (HMCs) induces βPix and p66Shc up-regulation, resulting in the formation of the βPix/p66Shc complex. In transformed HMCs, ET-1 induces a biphasic phosphorylation of p66Shc and FOXO3a. The second phase leads to p27kip1down-regulation independently of Akt. Depletion of βPix blocks the second phase of p66Shc and FOXO3a phosphorylation and prevents p27kip1down-regulation induced by ET-1. Depletion of either βPix or p66Shc inhibits ET-1–induced cell proliferation. The expression of β1Pix induces FOXO3a phosphorylation through activation of Rac1, ERK1/2, and p66Shc. Using either p66Shc- or Akt-depleted cells; we show that β1Pix-induced FOXO3a phosphorylation requires p66Shc but not Akt. β1Pix-induced p27kip1down-regulation was blocked by U0126 but not by wortmannin. Endogenous βPix and FOXO3a are constitutively associated with endogenous p66Shc. FOXO3a and p66Shc binding requires β1Pix homodimerization. Expression of β1Pix homodimerization deficient mutant abrogates β1Pix-induced p27kip1down-regulation and cell proliferation. Our results identify p66Shc and FOXO3a as novel partners of β1Pix and represent the first direct evidence of β1Pix in cell proliferation via Erk/p66Shc-dependent and Akt-independent mechanisms.