Updated project metadata. Colorectal cancer is one of the most common and widespread disease in the world and the third type of cancer causing a high mortality rate. There is increasing evidence that some polyunsaturated fatty acids (PUFAs) are involved in the reduction of cancer risk and progression. Recent studies showed that sn-2 monoacylglycerols (MAG) exercise specific inhibitory actions on cancer cells through different mechanisms. However, the anticancer effect of PUFA-based MAGs on colorectal cancer has yet to be assessed. In this work we investigated the actions of two PUFAs, docosahexaenoic (DHA, 22: 6n3) and arachidonic acids (ARA, 20:4n6), both as MAG, on colon cancer human (HT-29) cell line. We performed the MTT test, LDH, and caspase-3 assays, while global proteome changes were assessed by SWATH-MS quantitative proteomics followed by pathway analysis in order to find out which molecular mechanisms were being affected. It has been proven that ARA- and DHA-MAG exercises dose- and time-dependent antiproliferative actions. In all cases, DHA-MAG acts on cancer cells more efficiently than ARA-MAG. Results clearly demonstrate the ability of MAGs to induce cell death in colon cancer cells and suggest a direct relationship between their chemical structure and their potency. Therefore, sn-2 MAG are suitable candidate for the production of new functional ingredients.