A series of rhein derivatives (4a-x) were synthesized and evaluated for their aqueous solubility and their in vitro anti-proliferative activities on six different tumor cell lines. The aqueous solubility of the compounds was in the range of 10.04 to 15.08 mg/mL, which is 220 to 330-fold higher than that of rhein (0.0456 mg/mL). All derivatives displayed more potent anti-tumor activity than rhein, and most of them were even stronger than 5-FU, particularly, 4s, 4t and 4v (IC50: 3.01-5.28 µM on A549 cells, 5.92-7.63 µM on Mcf-7, 0.33-0.85 µM on HepG2 cells, 0.31-0.83 µM on HCT116, 2.36-6.49 µM on Bel-7402 and 4.48-7.31µM on Bel-7402/5-FU cells). Compound 4v was further found to induce HCT116 cell apoptosis, and to induced cell cycle G2/M -phase arrest via downregulation of CDK1 and cyclin B in HCT116 cell. Our findings suggest that 4v could be a promising lead for future studies.