Nutlin-3 shows a potent antitumor efficacy through downregulation of the cancerogenic ether a go-go 1 (Eag1) channel. However, the molecular mechanisms responsible for the regulation of Eag1 by Nutlin-3 in cancer cells remain unclear. In this study, we propose a novel anticancer mechanism of Nutlin-3, in which Nutlin-3 acts through the p53-Eag1-PI3K/AKT pathway. We first confirmed that Eag1 was downregulated through the activation of p53 by Nutlin-3. We then revealed that the inhibition of Eagl electrophysiological function resulted in the decrease of viability, migration and invasion of HeLa cells. It is worth noting that the antitumor effect of Nutlin-3 was abolished in the Eagl knockdown HeLa cell lines by siRNA. And Nutlin-3 can decrease the cell viability of H8 cells which were stably transfected with Eag1, but has no obvious inhibitory effect on blank H8 cells. Finally, we demonstrated that the decrease in Eag1 channel activity induced by Nutlin-3 treatment exerts anticancer activity by inhibiting the PI3K/AKT pathway. Our study therefore fills the gap between p53 pathway and its cellular function mediated by Eag1, shedding light on the new anti-cancer mechanism of Nutlin-3. (C) 2019 Elsevier Inc. All rights reserved.