Carthamus tinctorius L. (Safflower) has been often preferred because of rich fatty acid, flavonoid, alkaloid, and polysaccharide contents in its different parts in medicine and industrial area. Although its antioxidant, antienflamatuar, and antitumor properties have been proven in many studies, the mechanism underlying the anticancer activity is still more unclear. This study was first conducted to elucidate the apoptotic gene expression changes in human colorectal (CaCo-2), lung (A549), and cervix cancer (HeLa) cells after exposure to safflower seed oil (SFO). Cytotoxic activity of cancer cells was evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazoliumbromide) assay and then, total RNA derived from cell lines to analyze the gene expression profile on Real-Time Ready Human Apoptosis Panel 96 was used. MTT results showed that SFO greatly inhibited A549, CaCo-2 and HeLa cell proliferation, with a value of IC50 of 1.26, 3.92 and 13.12 mu g/ml, respectively. According to the cDNA microarray analysis, 56 genes were interpreted in connection with extrinsic, intrinsic, PI3K/AKT, JAK/STAT, and NF kappa B pathways. SFO treatments triggered apoptosis through the caspase-dependently pathway along with upregulated the expressions of many pro-apoptotic genes in the extrinsic and intrinsic pathway in HeLa cells. However, in A549 and CaCo-2 cells, SFO treatments were inhibited cell survival mechanism through frequently caspase-independent genes following downregulated the expression of anti-apoptotic genes. It is noteworthy that although cancer cells have different sensitivity, SFO induced apoptosis through different pathways. Taken together, SFO, as a natural resource, has the potential to be used as a promising agent against cancer, especially in gene therapy level.