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Purpose: Many athletes are deficient in long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA). A consequent low Omega-3 Index (O3I) and high arachidonic acid/eicosapentaenoic acid (AA/EPA) ratio increase cardiovascular disease risk and inflammation. Algae oil is a plant-based, sustainable source of LC n-3 PUFA, suitable for vegans and vegetarians. Effects of algae oil supplementation on whole blood fatty acids among athletes has not been previously reported. This study evaluated the effects of 5 weeks of DHA-rich algae oil supplementation on the whole blood fatty acid profile, O3I and AA/EPA ratio of omnivorous Division I American College Football (ACF) players. Methods: Data, including a spot blood sample, were collected at baseline for all participants (n = 47), then for a subset of players (n = 22) following a 5-week control period (usual diet) and 5 weeks of algae oil supplementation (usual diet + 1575 mg docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) 5 days/week; average 1125 mg/day). Results: Baseline O3I was 4.3% ± 0.1% and AA/EPA ratio was 45.6 ± 23.8. After 5 weeks of algae oil supplementation, the O3I was 6.1% ± 1.0% and the AA/EPA ratio was 25.1 ± 11.6. The O3I was significantly higher and the AA/EPA ratio was significantly lower (P < 0.0001 for both) compared with both baseline and the end of the control period. The increase in O3I from baseline was correlated with calculated DHA + EPA dose per unit body mass (R = 0.641, P = 0.001). Conclusions: Algae oil supplementation for 5 weeks improved both the low baseline O3I and high AA/EPA ratio among ACF players, with body mass specific dose effects.