Rationale The use of stable isotopes for ecological studies has increased exponentially in recent years. Isotopic trophic studies are based on the assumption that animals are what they eat plus a discrimination factor. The discrimination factor is affected by many variables and can be determined empirically. The Magellanic penguin is a highly abundant marine bird that plays a key role in the southern oceans. This study provides the first estimation of the Magellanic penguin blood discrimination factor for 13 C and 15 N. Methods A two and a half month feeding experiment was performed, in which ten captive penguins were fed their main natural prey (anchovy Engraulis anchoita ). The discrimination factors were estimated by comparing anchovy δ 13 C and δ 15 N values (obtained with isotope ratio mass spectrometry using lipid‐extracted and bulk anchovy muscle) with penguin blood δ 13 C and δ 15 N values. Results Penguin blood was shown to be enriched, compared with anchovies, for 13 C and 15 N. No changes were observed in the stable isotope ratios of anchovies and discrimination factors during the experiment. The overall discrimination factors were 0.93 ± 0.12 (bulk) and 0.41 ± 0.12 (lipid‐free) for 13 C; and 2.81 ± 0.17 (bulk) and 2.31 ± 0.17 (lipid‐free) for 15 N. Conclusions Having an accurate discrimination factor for the studied species is key in any trophic or food web isotopic study. Comparisons of estimated diet‐to‐blood discrimination factors with published values of aquatic piscivore birds showed that the 13 C discrimination factor is particularly variable, and therefore ecologists should be cautious when using a surrogate value from other species. In this study, the Magellanic penguin discrimination factor of a tissue that does not require euthanasia was obtained, a fundamental input for trophic isotopic modeling of the species. Copyright © 2016 John Wiley & Sons, Ltd.