Colon cancer is the third most commonly diagnosed cancer in the world. Research showed that arachidonic acid, a downstream ω-6 fatty acid (ω-6), plays a role in colon cancer development by producing deleterious metabolites from its COX-2 catalyzed peroxidation. On the other hand, dihomo-γ-linolenic acid (DGLA), the immediate precursor of arachidonic acid, may represent an exceptional ω-6 associated with anti-cancer activities. However, the mechanism of DGLA’s anti-cancer effect still remains unclear, and the rapid conversion of DGLA to arachidonic acid in human body by delta-5 desaturase (D5D) greatly restricts DGLA’s availability. Recent work from Dr. Qian’s group demonstrated that DGLA can undergo a unique pathway during COX-2-catalyzed peroxidation and produce distinct free radical byproducts. Here we proposed that (1) DGLA’s anti-cancer activity is derived from its distinct byproduct, e.g., 8-hydroxyoctanoic acid (8-HOA), from COX-2-catalyzed peroxidation, and (2) by knocking down cellular D5D expression, we can take advantage of the commonly overexpressed COX-2 in cancer cells to promote 8-HOA formation, inhibit colon cancer growth and migration, and develop a novel cancer therapy and a paradigm shift concept in contrast to classic COX-2 inhibition strategy in cancer treatment. Our results showed that 8-HOA, at physiological concentrations, could suppress human colon cancer cell growth and migration, by serving as a histone deacetylase inhibitor and DNA damage agents. Data also showed that knocking down D5D in colon cancer cells promoted endogenous formation of 8-HOA to a threshold level which then inhibited cancer cell growth and migration. Consistent with the in vitro data, knocking down D5D in human colon cancer cell-derived mice xenograft tumors along with DGLA supplementation promoted endogenous formation of 8-HOA in vivo and significantly suppressed tumor growth. In addition, direct supplementation and endogenous formation of 8-HOA from COX-2 catalyzed DGLA peroxidation were found to enhance the efficacies of various chemotherapeutic drugs. In conclusion, we demonstrated that by taking advantage of commonly overexpressed COX-2 in cancer, D5D knockdown can promote the formation of 8-HOA from DGLA peroxidation to inhibit cancer growth and migration. Results from this work will lead us to develop a novel ω-6 based treatment strategy for colon cancer.