Our recent studies have shown that Src family kinases (SFKs) are important mediators of proliferation in renal proximal tubular cells. In this study, we elucidate the signaling mechanisms that mediate SFK regulation of cell proliferation and cycle protein expression, and identify the SFK member responsible for these responses in a mouse renal proximal tubular cell line (RPTC). Akt, a target of phosphoinositide-3-kinase (PI3K), and ERK1/2 were constitutively phosphorylated in RPTC cultured in the presence of serum. While treatment of cells with PP1, a specific SFK inhibitor, completely blocked phosphorylation of ERK1/2 and Akt, only inhibition of PI3K/Akt resulted in decreased RPTC proliferation. Incubation of cells with PP1 decreased cyclin D1 expression, decreased p27 and p57 phosphorylation and increased p27 and p57 expression, two cyclin-dependent kinase inhibitors. Inhibition of the PI3K pathway decreased expression of cyclin D1 without altering expression of p27 and p57. In contrast, PP1 and PI3K inhibition had no effect on cyclin E and p21. Although RPTC expressed Src, Fyn and Lyn, only siRNA-mediated knockdown of Src decreased RPTC proliferation, decreased cyclin D1 expression, and increased p27 and p57 expression. These data reveal that Src is a crucial mediator of RPTC proliferation and Src-mediated proliferation is associated with PI3K-dependent up-regulation of cyclin D1 and PI3K-independent down-regulation of p27 and p57.