The human IL-3 receptor is composed of both α and β subunits. In early studies, we showed that the level of IL-3Rβ expression was lower in patients with acute promyelocytic leukemia (APL) than healthy donors and patients in complete remission by real-time quantitative polymerase chain reaction (RT-qPCR). With the differentiation of cells, enhanced expression of IL-3Rβ was also observed in all-trans-retinoic acid (ATRA)-induced NB4 cells. To unravel the role of IL-3Rβ upregulation in NB4 cells induced with ATRA, we knocked down IL-3Rβ expression by RNA interference (RNAi). Knockdown of IL-3Rβ resulted in decreased proliferation in NB4 cells induced with or without ATRA, observed by cell growth curves, colony formation assays and cell cycle analysis. Surface expression of CD11b antigen and nitroblue tetrazolium (NBT) reduction assays were also carried out at different time points. However, no significant difference was observed between the experimental and control groups treated with ATRA. Other findings suggested that IL-3Rα was decreased in NB4-IL-3Rβ shRNA cells by western blot. Down-regulation of IL-3Rβ also caused a decrease in PML/RARα expression detected with RT-qPCR. Together, these results suggest that abnormalities of IL-3Rβ expression were observed in APL; knockdown of IL-3Rβ inhibited the proliferation of NB4 cells with or without ATRA, but no effect was detected in the cellular differentiation. When NB4 cells exposed to ATAR, the up-regulation of IL-3Rβ expression may contribute to the maintenance of proliferation rather than cell differentiation.