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Currently, multi-drug resistance (MDR) to antineoplastic drugs is a major obstacle to successful treatment of carcinoma. Looking for novel agents with anti-MDR activity is an effectively way to overcome cancer drug resistance. Our previous study showed that podophyllotoxin derivative exhibited potent anti-proliferative effect and down-regulated the expression level of P-gp in K562/ADR cells, which probably was related with the MAPK pathways. However, the relation of P-gp expression and MAPK pathways still remains unclear. In this study, a series of aromatic heterocyclic esters of podophyllotoxin were synthesized and their anticancer effects were evaluated against two human chronic myeloid leukemia cell lines (K562 and K562/ADR), simultaneously, the initial structure-activity relationship was summarized. The most potent compound, Z5, displayed an IC50 value of 0.032 ± 0.006 μM against K562/ADR cells, with a lower resistant factor value of 1.280. Treatment of K562/ADR cells with Z5 caused S cell cycle arrest through reductions in cyclinA, cyclinB1, CDK1 and CDK2 levels. Moreover, Z5 treatment resulted in the induction of apoptosis as characterized by DNA staining, flow cytometry analysis and cleavage of procaspases-3, -8, -9 and PARP. Notably, Z5 significantly inhibited P-gp expression in K562/ADR cells. Additionally, Z5 also caused reactive oxygen species (ROS) generation, which was further demonstrated by preincubation with the antioxidant N-acetylcysteine (NAC). Western blotting revealed that Z5 markedly stimulated the MAPK pathways, including ERK1/2, JNK and P38, however, the mechanisms were prevented by NAC. Finally, the employment of NAC and MAPK inhibitors (U0126, SP600125 and SB203580) remarkably blocked the S phase arrest, apoptosis and down-regulation of P-gp induced by Z5. Taken together, Z5 strongly possessed the potential anti-MDR activity in K562/ADR cells through ROS/MAPK pathways-dependent S phase arrest, apoptosis and down-regulation of P-gp expression.