α-Linolenic acid (ALA, 18:3n-3) and γ-gamma linolenic acid (GLA, 18:3n-6) are polyunsaturated fatty acids (PUFA) that improve the human health. The present study focused on testing the in vitro antitumor actions of pure ALA and GLA on the HT-29 human colorectal cancer cell line. Cell viability was checked by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, cell membrane damage by the lactate dehydrogenase assay, apoptosis was tested by both caspase-3 activity trial and transmission electron microscopy images, and protein composition was analyzed by quantitative proteomics analysis. MTT test revealed IC₅₀ values of 230 and 255 μM for ALA and GLA, respectively, at 72 h. After 24 h of incubation, both ALA and GLA induced apoptosis on HT-29 colorectal cancer cells according to the caspase-3 assay and microscopy images. SWATH/MS analysis evidenced that ALA significantly affected the mitochondrial protein import pathway and the citric acid cycle pathway, while GLA did not significantly affect any particular pathway. In summary, both ALA and GLA showed concentration-dependent inhibitory effects on HT-29 cells viability and induced cell death by apoptosis. ALA significantly affected cellular pathways, while GLA does not have specific actions on either pathway. Both n-3 and n-6 C18 PUFA are bioactive food components useful in the colorectal cancer prevention.