To examine the effect of different omega-3 polyunsaturated fatty acid (n−3 PUFA) doses on the long chain (LC) n−3 PUFA enrichment in the egg yolk, Isochrysis galbana was supplemented in nine different doses (from 0 to 400mg n−3 PUFAs per 100g feed) to the diet of laying hens. A linear increase of the n−3 LC-PUFA enrichment was observed up to a dose of 120mg n−3 PUFAs per 100g feed. Higher n−3 PUFA supplementation doses resulted in a decreased efficiency of n−3 LC-PUFA incorporation into the egg yolk. Pronounced color changes were observed due to transfer of carotenoids from the microalgal biomass to the egg yolk. However, a corresponding linear trend in yolk color changes was not observed for the low supplementation doses when compared to the n−3 LC-PUFA changes in the egg yolk. The a*-value increased remarkably with supplementation of 3% Isochrysis (150mg n−3 PUFAs per 100g feed) and remained stable with the higher supplementation doses.