There is little EPA or DHA present in the yolks of eggs produced by hens fed corn-soybean meal-based diets. The purpose of this experiment was to enrich these n-3 fatty acids in the egg yolk by feeding hens with defatted green microalgae. A total of 50 White Leghorn hens (46-wk old, 1.70 ± 0.27 kg BW) were housed individually in cages, divided into 5 groups (n = 10), and fed a corn-soybean meal-based diet containing 0, 2.9, 5.8, 11.5, or 23% of a new type of defatted green microalgae (Nannochloropsis oceanica, Cellana, Kailua-Kona, HI) for 6 wk. Data were analyzed by one-way ANOVA with or without time-repeated measurements (SAS Version 9.1, SAS Institute, Inc., Cary, NC). The P-values for multiple regression analyses were adjusted using a Bonferroni correction procedure (significance at P ≤ 0.01). The microalgae supplementation produced dose-dependent linear (P < 0.01) enrichments of EPA, DHA, and total n-3 fatty acids consistently in egg yolk (R² > 0.9) assayed biweekly and of DHA in Liver (R² = 0.78), breast (R² = 0.82), and thigh (R² = 0.66) assayed at wk 6. Concentrations of EPA + DHA in egg yolk, liver, breast muscle, and thigh muscle of hens fed 11.5 and 23% microalgae were 1.4–2.1, 0.6–1, 3.3–5.3, and 6–7-fold greater (P < 0.001) than those in the controls (0% microalgae), respectively. Supplemental microalgae showed no effect on body weight, egg production rate, or tibia dimensions, but produced linear increases (P < 0.05) in tibia bone strength (R² = 0.71), egg shell weight (R² = 0.58), and egg yolk redness (R² = 0.71) and linear decreases (P < 0.05) in ADFI (R² = 0.88), egg yolk lightness (R² = 0.97), and egg yolk yellowness (R² = 0.80). In conclusion, this new type of defatted green microalgae was very effective in elevating EPA and DHA in the egg yolk, liver, and muscle tissues of layer hens. Eggs produced by hens fed the 23% defatted green microalgae diet may serve as a major food source of EPA and DHA to meet human needs. Supported in part by USDA/DOE Biomass R&D Initiative Grant, a Hatch Grant of Cornell University, and a Hatch Grant Supplement from Cornell University for Undergraduate Research.