Because the phosphatidylinositol-3-kinase-AKT pathway is emerging as an important regulator of tumor cell survival, inhibitors of this pathway have enormous potential in cancer treatment. A specific inhibitor of AKT, [d-3-deoxy-2-O-methyl-myo-inositol-1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate]] (SH-5) has been recently synthesized, but little is known about its effects on cytokine signaling. We found that SH-5 potentiated the apoptosis induced by tumor necrosis factor (TNF), as indicated by intracellular esterase staining, annexin V staining, and caspase-3 activation. This effect of SH-5 correlated with downregulation of various gene products that mediate cell survival, proliferation, metastasis, and invasion, all known to be regulated by NF-kappaB. SH-5 also blocked NF-kappaB activation induced by TNF-alpha, lipopolysaccharide, phorbol ester, and cigarette smoke but not that activated by hydrogen peroxide and RANK ligand, indicating differential requirement of AKT. Inhibition of NF-kappaB correlated with abrogation of phosphorylation and degradation of IkappaBalpha through the inhibition of activation of IkappaBalpha kinase (IKK). This led to suppression of the phosphorylation and translocation of p65 and also of NF-kappaB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, and IKKbeta but not that induced by p65 transfection. Thus, our results clearly demonstrate that inhibition of AKT leads to potentiation of apoptosis through modulation of NF-kappaB signaling.