Cantharidin is a natural toxin that possesses potent anti-tumor properties. Its clinical application, however, is limited due to severe side-effects. Its cytotoxicity is believed to be mediated by the inhibition of serine/threonine protein phosphatase 2A. In order to identify new compounds with potential clinical therapeutic use, a series of cantharidin analogues, including those with skeletal modifications at 1-C position (analogues 1-6) and those with anhydride modifications (analogues 7-13), were synthesized, and tested for their inhibitory effects on protein phosphatase 2A and their cytotoxicity to a panel of cancer cell lines. In addition, the mode of inhibition of cantharidin and analogue 13 on protein phosphatase 2A was determined by enzymatic kinetics assay. The data indicated that analogue 13 exhibited potent cytotoxicity to all cancer cell lines, and analogues 9, 11 and 12 showed relatively weak cytotoxicity to one or more cell lines, while other analogues showed little cytotoxicity. Accordingly, analogue 13 exhibited potent inhibitory activity on protein phosphatase 2A, and analogues 9, 11 and 12 showed weak inhibitory activity, while other analogues did not show any inhibitory activity. The findings indicate that the cytotoxicity of synthetic cantharidin analogues is likely to be associated with their protein phosphatase 2A inhibitory activity. The mode of inhibition of cantharidin and analogue 13 on protein phosphatase 2A is identified as noncompetitive inhibition by the Lineweaver-Burk plot.