BACKGROUND: The role of 1,25-dihydroxyvitamin D3 (1,25-(OH)(2)D(3)) in cancer prevention and treatment is an emerging topic of interest. However, its effects on the stemness of acute myeloid leukemia (AML) cells are poorly understood. METHODS: The proliferation and differentiation of AML cells (HL60 and NB4) were investigated by the CCK-8 assay, immunocytochemical staining, and flow cytometry. The abilities of HL60 and NB4 cells to form spheres were examined by the cell sphere formation assay. In addition, the levels of stemness-associated markers (SOX2, Nanog, OCT4, and c-Myc) in HL60 and NB4 cells were measured by western blotting and quantitative real-time polymerase chain reaction. Moreover, we obtained β-catenin-interacting protein 1 (ICAT)-knockout and ICAT-overexpressing HL-60 cells using gene editing and lentiviral infection techniques and investigated the role of ICAT in modulating the stemness-inhibiting effects of 1,25-(OH)(2)D(3) using the aforementioned experimental methods. Finally, we validated our findings in vivo using NOD/SCID mice. RESULTS: 1,25-(OH)(2)D(3) inhibited the proliferation and stemness of AML cells (HL60 and NB4) and induced their differentiation into monocytes. Additionally, the knockdown of ICAT in HL60 cells attenuated the inhibitory effects of 1,25-(OH)(2)D(3) on proliferation and stemness and suppressed the expression of stemness markers. Conversely, overexpression of ICAT enhanced the aforementioned inhibitory effects of 1,25-(OH)(2)D(3). Consistently, in NOD/SCID mice, 1,25-(OH)(2)D(3) suppressed tumor formation by HL-60 cells, and the effects of ICAT knockdown or overexpression on 1,25-(OH)(2)D(3) aligned with the in vitro findings. CONCLUSION: 1,25-(OH)(2)D(3) inhibits AML cell stemness, possibly through modulation of the ICAT-mediated Wnt/β-catenin signaling pathway.