Two PUFA, docosahexaenoic (DHA, 22: 6n3) and arachidonic acids (ARA, 20: 4n6), as well as their derivatives such as eicosanoids, regulate different activities, which include changes in receptor signaling, the composition of rafts, cell metabolism, and membrane structures. These also modify the function of transcription factors and their target genes, a key step essential for the function of FA-activated signaling. This work is focused to determine the antitumor actions of these compounds linked to the regulation of gene transcription on HT-29 colorectal cancer. For this, we performed antiproliferative antitumour assay, LDH breakage test, caspase-3 production, and proteome changes were assessed by SWATH-MS quantitative proteomics followed by pathway analysis in order to find out which molecular mechanisms were being affected. In all cases tested, DHA exercised antitumor actions to a higher extent than ARA, acting mainly by means of down-regulating most of the proteasome system particles, while ARA presented a heavy effect on all the six DNA replication helicase particles but did not affect proteasome. The results indicated that both DHA and ARA stopped colorectal cancer growth and proliferation, thus they represent the ideal candidates as chemopreventive agents.