BACKGROUND: Breast cancer is the most prevalent disease and the fourth cause of cancer death among female globally. The N6-methyladenylate methylation (m(6)A) demethylase alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) decreases modification of RNA, while its role in regulating breast cancer development remains unclear. METHODS: ALKBH5-silenced breast cancer cell-line MCF-7 was constructed to investigate its functional impact. Cell proliferation, migration and invasion ability were evaluated by CCK8 and transwell assays under ALKBH5 inhibition. Spheroid formation and in vitro extreme limiting dilution analysis (ELDA) were performed to elucidate the effect of ALKBH5 deficiency on stemness of MCF-7 cells. The m(6)A modification level of CTNNB1 and the interaction of ALKBH5 and CTNNB1 were investigated by Methylated RNA immunoprecipitation (MeRIP) and RIP assay respectively. RESULTS: Silencing ALKBH5 significantly suppressed MCF-7 cell proliferation, migration, and invasion abilities. Moreover, ALKBH5 depletion also diminished the stemness of breast cancer cells in vitro. Further investigation illustrated that ALKBH5 may regulate Wnt/β-catenin signaling via an m(6)A-dependant manner. Clinical data analysis demonstrated a strong positive relationship between ALKBH5 and β-catenin expression. CONCLUSION: This study establishes a link between ALKBH5 and cancer stemness in breast cancer, providing insights into the functional role of demethylase ALKBH5 in breast cancer progression.