Using the doxorubicin-sensitive K562 cell line and the resistant derivative lines KD30 and KD225 as models, we found that acquisition of multidrug resistance (MDR) is associated with enhanced FOXO3a activity and expression of ABCB1 (MDR1), a plasma membrane P-glycoprotein that functions as an efflux pump for various anticancer agents. Furthermore, induction of ABCB1 mRNA expression on doxorubicin treatment of naive K562 cells was also accompanied by increased FOXO3a activity. Analysis of transfected K562, KD30, and KD225 cells in which FOXO3a activity can be induced by 4-hydroxytamoxifen showed that FOXO3a up-regulates ABCB1 expression at protein, mRNA, and gene promoter levels. Conversely, silencing of endogenous FOXO3a expression in KD225 cells inhibited the expression of this transport protein. Promoter analysis and chromatin immunoprecipitation assays showed that FOXO3a regulation of ABCB1 expression involves binding of this transcription factor to the proximal promoter region. Moreover, activation of FOXO3a increased ABCB1 drug efflux potential in KD30 cells, whereas silencing of FOXO3a by siRNA significantly reduced ABCB1 drug efflux ability. Together, these findings suggest a novel mechanism that can contribute towards MDR, involving FOXO3a as sensor for the cytotoxic stress induced by anticancer drugs. Although FOXO3a may initially trigger a program of cell cycle arrest and cell death in response to doxorubicin, sustained FOXO3a activation promotes drug resistance and survival of cells by activating ABCB1 expression.