Abstract The side population cells characterized by the ability to transport Hoechst 33342 out of cells have been identified as cancer stem-like cells. ABCG2 is found to confer the side population (SP) phenotype, multidrug resistance (MDR) and tumor recurrence. In this study, we found secalonic acid D (SAD), a metabolite of marine-derived mangrove endophytic fungus, showed potent anticancer effect on ABCB1-, ABCC1- and ABCG2- overexpressing multidrug resistance cells by MTT assay. Furthermore, SAD could down-regulate the expression of ABCG2 and decrease the percentage of SP cells in lung cancer cells. The MTT assay showed the sorted SP cells were sensitive to SAD and we also found SAD could inhibit the sphere-forming ability of SP cells. Although SAD did not affect the expression of ABCG2 mRNA, it shortened the half-life of ABCG2 protein by activating calpain 1. These results implicated that SAD could be developed as a leading compound to target cancer stem cells and would be a promising agent to treat lung cancer patients.