In previous work, we presented experimental and theoretical evidence that podophyllum derivatives substituted by chlorine atom in the 3-posititon of 2-aminopyridine exhibited significantly elevated potency. In this study, a series of podophyllum derivatives substituted in the 3-position of 2-aminopyridine, including methyl and fluorine groups, were synthesized. Their chemical structures were confirmed by the spectral (H-1-nuclear magnetic resonance, C-13-nuclear magnetic resonance, electrospray ionization mass spectrometry) and elemental analyses. These derivatives were tested for their respective cytotoxicities in HeLa, BGC-823, A549, Huh7, and MCF-7 cells by MTT assay and the pharmacological results showed that most of them displayed potent cytotoxicities against at least one of the tested cancer cell lines. Structure-activity relationship study suggested that the introduction of the fluorine atom into the 3-posititon of 2-aminopyridine had enhanced the cytotoxicity against numerous tumor cells compared to the chlorine atom, while the methyl group did not. Furthermore, other biological experiments were consistent with the beneficial effect of fluorine atom substituent in the 3-position of 2-aminopyridine, which then inhibited the microtubule polymerization and activity of topoisomerase II when 2-amino-3-fluoropyridine substituted in podophyllotoxin and 4'-O-demethylepipodophyllotoxin, and that they work by effecting the target proteins which induce P53-dependent apoptosis.