Breast cancer is the leading cause of cancer death in the female population, despite advances in diagnosis and treatment. The highly heterogeneous nature of the disease represents a major obstacle to successful therapy and results in a significant number of patients developing drug resistance and, eventually, suffering from tumor relapse. Cancer stem cells (CSCs) are a small subset of tumor cells characterized by self-renewal, increased tumor-initiation capacity, and resistance to conventional therapies. As such, they have been implicated in the etiology of tumor recurrence and have emerged as promising targets for the development of novel therapies. Here, we show that the histone demethylase lysine-specific demethylase 1 (LSD1) plays an important role in the chemoresistance of breast cancer cells. Our data, from a series of in vitro and in vivo assays, advocate for LSD1 being critical in maintaining a pool of tumor-initiating cells that may contribute to the development of drug resistance. Combinatory administration of LSD1 inhibitors and anti-cancer drugs is more efficacious than monotherapy alone in eliminating all tumor cells in a 3D spheroid system. In conclusion, we provide compelling evidence that LSD1 is a key regulator of breast cancer stemness and a potential target for the design of future combination therapies.Keywords: KDM1A; breast cancer stem cells; combination therapy; lysine-specific demethylase 1; self-renewal; therapy resistance; tumor heterogeneity; tumor initiation.