Some polyunsaturated fatty acids (PUFAs), if not all, have been shown to have tumoricidal action, but their exact mechanism(s) of action is not clear. In the present study, we observed that n-6 PUFA linoleic acid (LA) inhibited tumor cell growth at high concentrations (above 300 μM); while low concentrations (100-200 μM) promoted proliferation. Analysis of cell mitochondrial membrane potential, reactive oxygen species (ROS) formation, malondialdehyde (MDA) accumulation and superoxide dismutase (SOD) activity suggested that anti-cancer action of LA is due to enhanced ROS generation and decreased cell anti-oxidant capacity that resulted in mitochondrial damage. Of the three cell lines tested, semi-differentiated colorectal cancer cells RKO were most sensitive to the cytotoxic action of LA, followed by undifferentiated colorectal cancer cell line (LOVO) while the normal human umbilical vein endothelial cells (HUVEC) were the most resistant (the degree of sensitivity to LA is as follows: RKO > LOVO > HUVEC). LA induced cell death was primed by mitochondrial apoptotic pathway. Pre-incubation of cancer cells with 100 μM LA for 24 hr enhanced sensitivity of differentiated and semi-differentiated cells to the subsequent exposure to LA. The relative resistance of LOVO cells to the cytotoxic action of LA is due to a reduction in the activation of caspase-3. Thus, LA induced cancer cell apoptosis by enhancing cellular oxidant status and inducing mitochondrial dysfunction.