Abstract: To explore an efficient strategy for the transformation of antibacterial fluoroquinoles into antitumor fluoroquinolones and their structure-activity relationship, an acylhydrazone as bioisostere of the C-3 carboxylic group, twelve novel fluoroquinolone C-3 acylhydrazones 3a - 3l were synthesized from ciprofloxacin, respectively. The structures were characterized by element analysis and spectral data. The in vitro antitumor activity against SMMC- 7721, L1210 and HL60 cell lines exhibited more significantly inhibitory activity than the parent, in which compounds with electron-withdrawing group were comparable to doxorubicin. SAR showed that compounds with electron-withdrawing group had more potency than those with electron-donating group, after reduction of acylhydrazone moiety, antitumor activity disappeared. Thus, it is necessary for an acylhydrazone as a bioisostere of the C-3 carboxylic group to develop antitumor fluoroquinolone lead compounds.