A series of N-phenyl substituted isatin derivatives were designed and synthesized about 40-80% overall yields. Three of the eleven newly synthesized compounds, compound 9, 10 and 11, have not been reported before. Their structures were characterized by 1H and 13C NMR. All the newly synthesized derivatives were subjected to evaluate their cytotoxic properties against three human tumor cell lines, HepG2, HT-29 and K562. Results indicated that compounds 2, 3, 5, 8, 9 and 10 exhibited significant anti-tumor activities against liver cancer (HepG2), colon cancer (HT-29) and leukemia (K562) cell lines. Among, compound 9 with the IC50 values of 24.09 μM, 20.27 μM, 6.10 μM against cancer cell lines HepG2, HT-29 and K562.