A series of novel conjugates of podophyllotoxin and 5-FU were designed using association strategy and were synthesized by coupling 4'-demethylepipodophyllotoxin with 5-FU-N(1)-alkyl amino acid ester. These derivatives have been evaluated for cytotoxicity in vitro against tumour cell lines (HL-60, K562, A-549 and AGS), and their octanol-water partition coefficients (logP) were also determined. As compared with VP-16, most compounds showed superior water solubility, as well as more potent inhibitions against these four tumour cell lines. Compound 21 showed interaction with calf thymus DNA, and it was relatively resistant to metabolism by human plasma.