In a practical feeding trial at Ouwehand Zoo, plasma concentrations of vitamin A₁, calcidiol (D₃), α-tocopherol (E), and B₁ in 17 Humboldt penguins (Spheniscus humboldti) were measured before and after supplementation to gain insight into the effect of supplementing these vitamins in animals being fed thawed frozen-fish diets. None of the penguins received vitamin supplements for at least 6 mo before the supplementation trial, which was conducted prior to their normal nesting and molting period. During the trial period, eight penguins received daily vitamin A₁, D₃, tocopheryl acetate, and B₁ supplementation placed in their fish immediately prior to feeding and nine control penguins received no supplementation. Concentrations of vitamins A₁, D₃, α-tocopherol, and B₁ were also measured in the thawed ready-to-feed fish. Concentrations of vitamins B₁ and α-tocopherol were below the Association of Zoos and Aquariums (AZA) recommendations for penguin diets, while concentrations of vitamins A₁ and D₃ were far above AZA recommendations. At the start of the study and after 70 days of supplementation, plasma concentrations were determined for these vitamins. Vitamin B₁ concentrations in plasma increased significantly (P < 0.05) between Day 0 (mean 39.9 μg/L) and day 70 (mean 160.5 μg/L) in the supplemented group. Plasma vitamin D₃ and α-tocopherol did not show a significant change. Vitamin A₁ levels in the supplemented group decreased significantly from 1.65 mg/L on day 0 to 1.4 mg/L on day 70. In the control group no significant changes were observed. The results of the study support the necessity of supplementing vitamin B₁ in penguins fed thawed frozen fish. Depletion of vitamin A and E concentrations in frozen food fish over time support recommendations to regularly measure vitamin concentrations in different batches of frozen fish.