Astaxanthin from a transgenic maize line was evaluated as feed supplement source conferring effective pigmentation of rainbow trout flesh. An extraction procedure using ethanol together with the addition of vegetal oil was established. This resulted in an oily astaxanthin preparation which was not sufficiently concentrated for direct application to the feed. Therefore, a concentration process involving multiple phase partitioning steps was implemented to remove 90% of the oil. The resulting astaxanthin raw material contained non esterified astaxanthin with 12% 4-keto zeaxanthin and 2% zeaxanthin as additionalcarotenoids. Isomeric analysis confirmed the exclusive presence of the 3S,3’S astaxanthin enantiomer. The geometrical isomers were 89% all-E, 8% 13-Z and 3% 9-Z. The incorporation of the oily astaxanthin preparation into trout feed was performed to deliver 12 mg/kg astaxanthin in the final feed formulation for the first 3.5 weeks and 72 mg/kg for the final 3.5 weeks of the feeding trial. The resulting pigmentation of the trout fillets was determined by hue values with a colour meter and further confirmed by astaxanthin quantification. Pigmentation properties of the maize-produced natural astaxanthin incorporated to 3.5 μg/g dw in the trout fillet resembles that of chemically synthesized astaxanthin. By comparing the relative carotenoid compositions in feed, flesh and feces, a preferential uptake of zeaxanthin and 4-keto zeaxanthin over astaxanthin was observed.