Many species of seabirds, including the only penguin species breeding on the African continent, are threatened with extinction. The world population of the endangered African penguin Spheniscus demersus has decreased from more than 1.5 million individuals in the early 1900s to c.a. 23 000 pairs in 2013. Determining the trophic interactions of species, especially those of conservation concern, is important when declining numbers are thought to be driven by food limitation. By and large, African penguin dietary studies have relied on the identification of prey remains from stomach contents. Despite all the advantages of this method, it has well known biases. We therefore assessed the African penguin's diet, using stable isotopes, at two colonies in Algoa Bay (south-east coast of South Africa). These represent over 50% of the world population. Various samples (blood, feathers, egg membranes) were collected for carbon and nitrogen stable isotope analyses. Results indicate that the trophic ecology of African penguins is influenced by colony, season and age class, but not adult sex. Isotopic niches identified by standard Bayesian ellipse areas and convex hulls, highlighted differences among groups and variability among individual penguins. Using Bayesian mixing models it was for the first time shown that adults target chokka squid Loligo reynaudii for self-provisioning during particular stages of their annual cycle, while concurrently feeding their chicks primarily with small pelagic fish. This has important ramifications and means that not only pelagic fish, but also squid stocks, need to be carefully managed in order to allow population recovery of African penguin.