The 40S ribosomal protein S6 kinase (S6K) acts downstream of mTOR, which plays important roles in cell proliferation, protein translation, and cell survival and is a target for cancer therapy. mTOR inhibitors are, however, of limited success. Although Akt is believed to act upstream of mTOR, persistent inhibition of p70 S6 kinase or S6K1 can activate Akt via a negative feedback loop. S6K exists as two homologues, S6K1 and S6K2, but little is known about the function of S6K2. In the present study, we have examined the effects of S6K2 on Akt activation and cell survival. Silencing of S6K1 caused a modest decrease, whereas knockdown of S6K2 caused a substantial increase in TNF-a and TRAIL (TNF-related apoptosis-inducing ligand)-mediated apoptosis. In contrast to S6K1, depletion of S6K2 by siRNA decreased basal and TNF-induced Akt phosphorylation. Ectopic expression of constitutively active Akt in MCF-7 cells restored cell survival in S6K2-depleted cells. We have previously shown that activation of Akt induces downregulation of Bid via p53. Knockdown of S6K2 caused an increase in p53, and downregulation of p53 by siRNA decreased Bid level. Silencing of Bid blunted the ability of S6K2 deficiency to enhance TNF-induced apoptosis. Taken together, our study shows that the two homologues of S6K have distinct effects on Akt activation and cell survival. Thus, targeting S6K2 may be an effective therapeutic strategy to treat cancers.