Mel‐18 has been proposed as a negative regulator of Bmi‐1, a cancer stem cell (CSC) marker, but it is still unclear whether Mel‐18 is involved in CSC regulation. Here, we examined the effect of Mel‐18 on the stemness of human breast CSCs. In Mel‐18 small hairpin RNA (shRNA)‐transduced MCF‐7 cells, side population (SP) cells and breast CSC surface marker (CD44⁺/CD24^–/ESA⁺)‐expressing cells, which imply a CSC population, were enriched. Moreover, the self‐renewal of CSCs was enhanced by Mel‐18 knockdown, as measured by the ability for tumorsphere formation in vitro and tumor‐initiating capacity in vivo. Similarly, Mel‐18 overexpression inhibited the number and self‐renewal activity of breast CSCs in SK‐BR‐3 cells. Furthermore, our data showed that Mel‐18 blockade up‐regulated the expression of the Wnt/TCF target Jagged‐1, a Notch ligand, and consequently activated the Notch pathway. Pharmacologic inhibition of the Notch and Wnt pathways abrogated Mel‐18 knockdown‐mediated tumorsphere formation ability. Taken together, our findings suggest that Mel‐18 is a novel negative regulator of breast CSCs that inhibits the stem cell population and in vitro and in vivo self‐renewal through the inactivation of Wnt‐mediated Notch signaling.—Won, H.‐Y., Lee, J.‐Y., Shin, D.‐H., Park, J.‐H., Nam, J.‐S., Kim, H.‐C., Kong, G. Loss of Mel‐18 enhances breast cancer stem cell activity and tumorigenicity through activating Notch signaling mediated by the Wnt/TCF pathway. FASEB J. 26, 5002–5013 (2012). www.fasebj.org