Central role of constitutively active protein kinase B/Akt (PKB) in melanoma drives the search for new targets to abolish its deranged signaling. PKB activation is promoted by cholesterol-enriched lipid rafts and is Ca. 2+-dependent, but the pathway linking rafts and Ca2+ to deregulation of this enzyme remains poorly understood. Here employing B16BL6 melanoma model, we show that ablation of rafts with methyl-β-cyclodextrin (MβCD) inactivated PKB by inhibiting Src kinase and reactivating the negative PKB modulator, PP2A phosphatase. Blockade of PP2A with okadaic acid rescued PKB, indicating that raft ablation reactivated PP2A through inhibiting Src. Indeed, direct Src blockade with the Src kinase inhibitor-1 or the dominant-negative Src-mutant was sufficient for PP2A reactivation and downregulation of PKB, whereas reconstitution of rafts in MβCD-treated cells restored PKB, PP2A and Src activities to their basal levels. This pathway was also interrupted by inhibition of the Ca2+ sensor calmodulin, either by its antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide or the Ca2+-insensitive calmodulin-mutant or the intracellular Ca2+-chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N#N#-tetraacetic acid tetra-(acetocymethyl)-ester or by diminishing the store-operated Ca2+ entry with 2-aminoethoxydiphenyl borate or small hairpin RNA against Stim1. Ablation of rafts prevented Stim1-mediated store-operated Ca2+ entry, aborted Ca2+ stimulation of raft-residing calmodulin and disrupted its Ca2+-dependent binding to Src, abolishing Src activity and entire Src/PP2A/PKB cascade. Most importantly, blockade of this cascade in the tumor site by raft-ablating MβCD, administered to melanoma-bearing mice, robustly retarded tumor growth and extended animal survival. Together, our data suggest that lipid rafts couple store-operated Ca2+ entry to sustained activation of major tumor-promoting signaling elements in melanoma cells and underscore the potential of raft-targeting agents as effective anticancer drugs.