Background: A growing body of evidence suggests that alterations of dietary fatty acid (FA) profiles are associated with colorectal cancer (CRC) risk. However, data from large-scale epidemiological studies using circulating FA measurements to objectively assess individual FA and FA categories are scarce. Methods: To investigate the association between red blood cell (RBC) membrane FAs and risk of CRC in a case-control study nested within a large prospective cohort. After a median follow-up of 6.4 years, 1069 incident CRC cases were identified and matched to 1069 controls among participants of the European Prospective Investigation into Cancer and Nutrition (EPIC). The FA composition of RBC phospholipids (in mol%) was analyzed by gas chromatography, and their association with risk of CRC was estimated by multivariable adjusted conditional logistic regression models. Results: After correction for multiple testing, subjects with higher concentrations of RBC stearic acid were at higher risk for CRC (OR=1.23; 95% CI=1.07-1.42, per 1 mol%). Conversely, CRC incidence decreased with increasing proportions of RBC n-3 PUFA, particularly eicosapentaenoic acid (0.75; 0.62-0.92, per 1 mol%). The findings for the n-6 PUFA arachidonic acid were inconsistent. Conclusions: The positive association between pre-diagnostic RBC stearic acid and CRC reflects putative differences in FA intake and metabolism between cancer cases and matched controls which deserve further investigation. The inverse relationship between EPA and CRC is in line with the repeatedly reported protective effect of fish consumption on CRC risk. Impact: These findings add to the evidence on CRC prevention.