Abstract Evidence is emerging that Foxo family proteins serve as biochemical signal integrators in complex signaling networks mediating and modulating diverse cellular functions. Herein, we report that besides the well-established function of Foxo3a as a transcriptional regulator of multiple target genes in nucleus, a substantial fraction of Foxo3a translocates to mitochondria leading to disruption of mitochondrial membrane potential, release of cytochrome c and caspase activation during erythroid differentiation mediated by guanosine 5′-triphosphate (GTP). In fact, non transcriptional role of Foxo3a in mitochondria was achieved through the protein–protein interaction with pro-apoptotic protein Bax and its translocation to mitochondrial membrane. Furthermore, our results revealed that mitochondrial localization of Foxo3a was modulated by intracellular GTP content which is sensed by PKC signaling element. Collectively, our findings provided insight into a novel Foxo3a mechanism in leukemia cells which led to engagement of cells in the maturation pathway.