Norcantharidin (NCTD), a chemically modified form of cantharidin, is a potential anticancer drug. This study investigated the effect of NCTD on anoikis in CT26 colorectal adenocarcinoma cells. NCTD treatment of CT26 cells showed a dose-dependent and time-dependent decrease in viability and cell proliferation. Growth inhibition was accompanied by cell cycle arrest in the S and G2/M phases. Mitogen-activated protein kinase expression, assayed by Western blot, was unchanged except for Jun-N-terminal kinase (JNK). At 24 h of treatment with 0-20 micromol/l NCTD, JNK expression increased at 24 h, but then decreased at 48 h; in contrast, the phosphorylated JNK levels markedly increased. JNK inhibitor (SP600125) in the culture effectively blocked NCTD-induced cytotoxicity and detachment of cells. CT26 cells treated with NCTD not only displayed inhibited cell adhesion and down-expression of integrin beta1, but also changed from being shuttle-shaped to round, the latter cells being more susceptible to anoikis-mediated apoptosis. Flow cytometric assay of the DNA content in NCTD-treated CT26 cells at 24 and 48 h showed a marked increase in the sub-G1 level, indicating that NCTD induced apoptosis. NCTD inhibited the viability of CT26 cancer cells preferentially over normal bone marrow and mononuclear cells. NCTD inhibits CT26 cancer cells by blocking proliferation and inducing anoikis-mediated apoptosis, a process that might be regulated by JNK activation.