Obesity is characterized by low-grade chronic inflammation, which can be modulated by lipid mediators derived from omega-3 (n-3) polyunsaturated fatty acids (PUFA). Obesity is a multifactorial disease, where genetic and environmental factors strongly interact to increase its development. In this context, the FADS1 gene encodes the delta-5 desaturase protein, which catalyzes the desaturation of PUFA. The rs174547 genetic variant of FADS1 has been associated with alterations in lipid metabolism, particularly with decreases in eicosapentaenoic acid (EPA) and arachidonic acid (AA) concentrations. To analyze the effect of an n-3-supplemented diet on the fatty acid profile and composition in red blood cells (RBCs) of obese subjects carrying the rs174547 variant of the FADS1 gene. Seventy-six subjects with obesity were divided into two groups: omega-3 (1.5 g of n-3/day) and placebo (1.5 g of sunflower oil/day). The dietary intervention consisted of a four-month follow-up. Anthropometric, biochemical, and dietary variables were evaluated monthly. The total fatty acid profile in RBC was determined using gas chromatography. The rs174547 variant was analyzed through allelic discrimination. The n-3 index (O3I) increased at the end of the intervention in both groups. Subjects carrying the CC genotype showed significant differences (minor increase) in n-6, n-3, total PUFA, EPA, DHA, and the O3I in RBCs compared to TT genotype carriers in the n-3 group. The diet supplemented with EPA and DHA is ideal for providing the direct products that bypass the synthesis step affected by the FADS1 rs174547 variant in subjects carrying the CC genotype. The O3I confirmed an increase in n-3 fatty acids in RBCs at the end of the intervention.