We report herein the design and synthesis of a series of novel fluoride-containing gossypol derivatives by the condensation reaction of gossypol with fluoride-containing aromatic amine. These fluoride-containing gossypol derivatives were characterized by IR, ¹H-NMR and high resolution mass spectral data, then screened as antitumor agents against three human cancer cell lines (HeLa, A-549 and BGC-823) and a normal cell line (VEC) in vitro by using MTT cell proliferation assays. Results revealed that compounds 3a, 3c and 3f exhibited superior anticancer activity against HeLa, compounds 3b,3c, 3e and 3f exhibited superior anticancer activity against A-549, compounds 3b, 3c and 3f exhibited superior anticancer activity against BGC-823 compared to gossypol. In particular, fluorine substituent at the para positions of the phenyl ring showed remarkable inhibitory effects on HeLa (3c: IC₅₀ = 14.2 μM, 3f: IC₅₀ = 8.34 μM), A-549(3c: IC₅₀ = 6.32 μM, 3f: IC₅₀ = 9.76 μM) and BGC-823 cells (3c: IC₅₀ = 8.62 μM, 3f: IC₅₀ = 4.36 μM). Furthermore, all the compounds 3a-3f exhibited lessened cytotoxicity against VEC compared to gossypol.