Breast cancer is one of the most common cancer with high morbidity and mortality in women. This study aimed to explore the potential mechanism of costunolide inducing MCF‐7 cells apoptosis by multi‐spectroscopy, molecular docking, and cell experiments. The results manifested that costunolide interacted with calf thymus DNA (ct‐DNA) in a spontaneous manner, and the minor groove as the preferential binding mode. Furthermore, costunolide inhibited cell proliferation and colony formation. Hoechst 33258 staining showed that cell apoptosis induced by costunolide might be related to DNA damage. The apoptosis mechanism relied on regulating the protein expression of Bax, Bcl‐2, p53, Caspase‐3 and the activation of p38MAPK and nuclear factor κB (NF‐κB) pathways. This study will provide some experimental basis and potential therapeutic strategy for breast cancer treatment.