Chromosomal focal amplifications often cause an increase in gene copy number, contributing to the pathogenesis of cancer. PRR14 overexpression is associated with recurrent locus amplification in lung cancer, and it correlates with a poor prognosis. We show that increased PRR14 expression promoted and reduced PRR14 expression impeded lung cancer cell proliferation. Interestingly, PRR14 cells were markedly enlarged in size and exhibited an elevated activity of the PI3-kinase/Akt/mTOR pathway, which was associated with a heightened sensitivity to the inhibitors of PI3K and mammalian target of rapamycin (mTOR). Biochemical analysis revealed that PRR14, as a proline-rich protein, binds to the Src homology 3 (SH3) domains of GRB2 resulting in PI3K activation. Significantly, two cancer patient-derived PRR14 mutants displayed considerably augmented GRB2-binding and an enhanced ability of promoting cell proliferation. Together with the in vivo data demonstrating a strong tumor-promoting activity of PRR14 and the mutants, our work uncovered this proline-rich protein as a novel activator of the PI3K pathway that promoted tumorigenesis in lung cancer.