N-Myc downstream-regulated gene 1 (NDRG1) protein has been shown to be up-regulated during leukemic cell differentiation induced by some differentiation-inducing agents such as all-trans retinoic acid (ATRA). However, the potential role of up-regulated NDRG1 in the event is greatly unknown. In this work, we show that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while the knock-down of NDRG1 expression by specific small interfering RNA significantly antagonizes ATRA-induced differentiation of leukemic cells, proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, our work shows that CCAAT/enhancer-binding protein beta (C/EBPbeta) and PU.1, which are important hematopoiesis-related transcription factors, may act as downstream effectors of NDRG1 in leukemic cell differentiation. Taking together, this study provides direct evidence for the role of NDRG1 protein in myeloid leukemic cell differentiation.