Increased intake of n-3 PUFAs, such as docosahexaenoic acid (DHA;22:6n-3) could be beneficial in cancer therapy as it has been shown to exhibit several anti-cancer effects, including alteration of eicosanoid synthesis, regulation of gene expression, increased oxidative stress, induction of ER-stress, and affecting signalling pathways such as autophagy and apoptosis. It is possible that DHA could enhance the effects of chemotherapeutics used in treatment of colorectal cancer (CRC), including 5-fluorouracil (5-FU) and oxaliplatin (OX). The aim of this study was to investigate the effects of treatment with DHA, 5-FU and OX, both separately and combined and the molecular pathways involved, with a special focus on oxidative stress, autophagy and apoptosis. The two human colon cancer cell lines, DLD-1 and LS411N, grown in the same culturing media, responded differently to treatment with DHA and chemotherapeutics. The effect on cell growth was measured by cell counting and showed that DLD-1 cells were more sensitive to DHA, but less sensitive to 5-FU and OX, compared to LS411N cells. Co-treatment with DHA and 5-FU resulted in additional response in both cell lines, however more extensive in DLD-1 cells. The cell lines did not seem to have any additional effect from co-treatments with DHA and OX or with DHA combined with both 5-FU and OX. The XTT assay was used to measure cell viability but did not provide reliable information about the response to the treatments. Western blot was used to measure levels of proteins related to autophagy, oxidative stress and apoptosis. Confocal imaging was also used to investigate cellular levels and localization of autophagy- and oxidative stress-related proteins. Levels of the autophagy marker protein MAP1LC3B-II was upregulated in DLD-1 cells after treatment with DHA (35 µM) for 96 h, indicating a possible increase in autophagic flux by DHA treatment. Confocal imaging showed that the protein level of MAP1LC3B was higher in LS411N cells than DLD-1 cells, suggesting that the basal level of autophagy is important for DHA-sensitivity in different CRC cell lines. Both western blot and confocal imaging showed that levels of the autophagy-related protein SQSTM1 increased in both cell lines after treatment with DHA, both alone and in combination with chemotherapeutics. This may indicate that DHA could affect autophagy in the cell lines. The level of the oxidative stress-related protein NFE2L2 was also shown by confocal imaging to be increased in both cell lines after treatment with DHA, both alone and in combination with chemotherapeutics. This may indicate that DHA induces oxidative stress in both cell lines.