Cyclooxygenase (COX) can metabolize dihomo-γ-linolenic acid (DGLA) and arachidonic acid (AA) through free radical-mediated lipid peroxidation to form the anti-carcinogenic 1-series of prostaglandins and pro-carcinogenic 2-series of prostaglandins, respectively. Our previous studies had demonstrated that in ovine COX-mediated DGLA and AA peroxidation, there are common and exclusive free radicals formed through different free radical reactions. However, it was still unclear whether the differences are associated with the contrasting bioactivity of DGLA vs. AA. In order to investigate the possible association between cancer cell growth and the exclusive free radicals generated from COX/DGLA vs. COX/AA, we refined our combined spin-trapping/LC/MS method with solid phase extraction to characterize free radicals in their reduced forms in the human colon cancer cell line HCA-7 colony 29, which has a high COX-2 expression. For the first time, we were able to profile free radical formation in the experimental settings in which cell proliferation (via MTS assay) and cell cycle distribution (via PI staining) could be assessed. Our results showed that DGLA- and AA-derived exclusive free radicals, rather than prostaglandins, were closely associated with the opposing bioactivities of DGLA vs. AA. Due to rapid conversion from DGLA to AA via Δ-5 desaturase (D5D), the anti-proliferative effect of DGLA on cancer cell growth was limited. Thus, double doses of DGLA and D5D inhibitor were introduced. Among DGLA, double-dose DGLA, and combined DGLA/D5D inhibitor treatments, the latter exerted the most anti-proliferative effect on cancer cell growth and caused significant cell G2/M arrest. D5D knockdown cells (via siRNA transfection) were used to further investigate the possible mechanism underlying the anti-proliferative effect of DGLA on cancer cell growth. In addition, the combined DGLA/D5D Iv inhibitor treatment increased the susceptibility of cancer cells to the chemotherapy drug 5-fluorouracil. In D5D knockdown cells, DGLA and 5-fluorouracil exerted greater effects on cell growth inhibition and cytotoxicity due to synergism. In summary, increasing DGLA and concurrently decreasing AA in cells could be a novel approach to controlling the development of AA-dependent cancer. Our study allowed us to directly study the free radical-associated PUFA bioactivity, thus improving our understanding of COX-catalyzed lipid peroxidation in cancer biology.