Since the mTOR pathway is commonly deregulated in human cancer, mTOR inhibitors, rapamycin and its derivatives, are being actively tested in cancer clinical trials. Clinical updates indicate that the anti-cancer effect of these drugs is limited, perhaps due to rapamycin-dependent induction of oncogenic cascades by an, as yet, unclear mechanism. As such, we investigated rapamycin-dependent phosphoproteomics and discovered that 250 phosphosites in 161 cellular proteins were sensitive to rapamycin. Among these, rapamycin regulated 4 kinases and 4 phosphatases. A siRNA-dependent screen of these proteins showed that AKT induction by rapamycin was attenuated by depleting cellular CDC25B phosphatase. Rapamycin induces the phosphorylation of CDC25B at Serine375, and mutating this site to Alanine substantially reduced CDC25B phosphatase activity. Additionally, expression of CDC25B (S375A) inhibited the AKT activation by rapamycin indicating phosphorylation of CDC25B is critical for CDC25B activity and its ability to transduce rapamycin-induced oncogenic AKT activity. Importantly, we also found that CDC25B depletion in various cancer cell lines enhanced the anti-cancer effect of rapamycin. Together, using rapamycin phosphoproteomics, we not only advance the global mechanistic understanding of rapamycin’s action in cancer but also demonstrate that CDC25B may serve as a drug target for improving mTOR-targeted cancer therapies.