The presence of breast cancer stem cells (BCSCs) induces the aggressive progression and recurrence of breast cancer. These cells are drug resistant, have the capacity to self-renew and differentiate and are involved in recurrence and metastasis, suggesting that targeting BCSCs may improve treatment efficacy. In this report, methanol extracts of carrot root were purified by means of silica gel, Sephadex LH-20, and preparative high-performance liquid chromatography to isolate a compound targeting mammosphere formation. We isolated the compound 6-methoxymellein, which inhibits the proliferation and migration of breast cancer cells, reduces mammosphere growth, decreases the proportion of CD44⁺/CD24⁻ cells in breast cancer cells and decreases the expression of stemness-associated proteins c-Myc, Sox-2 and Oct4. 6-Methoxymellein reduces the nuclear localization of nuclear factor-κB (NF-κB) subunit p65 and p50. Subsequently, 6-methoxymellein decreases the mRNA transcription and secretion of IL-6 and IL-8. Our data suggest that 6-methoxymellein may be an anticancer agent that inhibits BCSCs via NF-κB/IL-6 and IL-8 regulation.