Three series of podophyllotoxin derivatives with various nitrogen-containing heterocycles were designed and synthesized. The antitumor activity of these podophyllotoxin derivatives was evaluatedin vitroagainst a panel of human tumor cell lines. The results showed that podophyllotoxin-imidazolium salts and podophyllotoxin-1,2,4-triazolium saltsa1–a20exhibited excellent cytotoxic activity. Among them,a6was the most potent cytotoxic compound with IC50values of 0.04–0.29 μM. Podophyllotoxin-1,2,3-triazole derivativesb1–b5displayed medium cytotoxic activity, and podophyllotoxin-amine compoundsc1–c3has good cytotoxic activity with IC50value of 0.04–0.58 μM. Furthermore, cell cycle and apoptosis experiments of compounda6were carried out and the results exhibited thata6could induce G2/M cell cycle arrest and apoptosis in HCT-116 cells.