The uptake of omega-3 polyunsaturated fatty acids (PUFAs) into the liver, gut mucosa, and tumor tissue and plasma levels after preoperative administration of supplemented enteral nutrition was investigated in patients with malignancies of the upper gastrointestinal tract. The objective of the study was to evaluate the incorporation of preoperatively administrated PUFAs, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) into cell phospholipids. Patients undergoing major gastrointestinal surgery (n = 40) were prospectively randomized to receive a PUFA-supplemented liquid oral diet 5 days preoperatively or an isocaloric control diet. The planned diet intake was 1000 mL/d providing 3.7 g of PUFA. The diet was given in addition to the usual hospital diet. The phospholipid fractions in plasma were analyzed on the day of surgery. Tissue samples of liver, gut mucosa (small intestine), and tumor were taken during surgery and homogenized. EPA and DHA content was analyzed using liquid gas chromatography. Both patient groups (PUFA group: n = 20; control group: n = 20) were similar in age, weight, and surgical procedures. As compared with the control group, the PUFA group had significantly increased levels of EPA in liver tissue (0.4 vs 1.3 weight %), gut mucosa (0.3 vs 1.0 weight %), and tumor tissue (0.3 vs 0.8 weight %). Also, the DHA levels in the PUFA group were significantly higher than the control group: liver tissue (4.1 vs 7.5 weight %), gut mucosa (2.1 vs 3.7 weight %) and tumor tissue (1.9 vs 4.2 weight %). This study suggests that administration of PUFA-enriched diets leads to increased incorporation of EPA and DHA not only in liver and gut mucosa tissue, but also in tumor tissue in patients with solid gastrointestinal tumors. Thus, preoperative administration of oral PUFA-enriched diets could have an impact on the postoperative inflammatory response after major abdominal surgery.