Abstract Background: A growing body of evidence suggests that alterations of dietary fatty acid (FA) profiles are associated with colorectal cancer risk. However, data from large-scale epidemiologic studies using circulating FA measurements to objectively assess individual FA and FA categories are scarce. Methods: We investigate the association between red blood cell (RBC) membrane FAs and risk of colorectal cancer in a case–control study nested within a large prospective cohort. After a median follow-up of 6.4 years, 1,069 incident colorectal cancer cases were identified and matched to 1,069 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 colorectal cancer 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 colorectal cancer (OR = 1.23; 95% CI = 1.07–1.42, per 1 mol%). Conversely, colorectal cancer 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 prediagnostic RBC stearic acid and colorectal cancer reflects putative differences in FA intake and metabolism between cancer cases and matched controls, which deserve further investigation. The inverse relationship between EPA and colorectal cancer is in line with the repeatedly reported protective effect of fish consumption on colorectal cancer risk. Impact: These findings add to the evidence on colorectal cancer prevention.