Hormones regulate many aspects of an individual's phenotype, including various physiological and behavioral traits. Two hormones have been described as important players in the regulation of parental investment in birds: the glucocorticoid hormone corticosterone and prolactin, a pituitary hormone, widely involved in mediating parental behavior. In comparison with corticosterone, the role of prolactin on parental investment remains poorly documented, and most studies so far have been correlative. In this study, the effects of an experimental decrease of prolactin levels on the incubation behavior of a long-lived seabird species were assessed. Male Adelie penguins were treated with self-degradable bromocriptine pellets, inhibiting prolactin secretion. Filming and subsequent video analysis allowed the determination of a behavioral time budget for birds and their position on the nest, while dummy eggs recorded incubation parameters. Incubation duration and breeding success at hatching were also monitored. As expected, bromocriptine-treatment significantly decreased plasma prolactin levels, but did not affect corticosterone levels. The behavioral time budget of penguins was not affected by the treatment. However, treated birds spent significantly more time in an upright position on the nest. These birds also incubated their eggs at lower temperatures and turned their eggs more frequently than controls, resulting in a lengthened incubation period. Despite this, the treatment was insufficient to trigger nest desertion and eggs of treated birds still hatched, indicating that several endocrine signals are required for the induction of nest abandonment. We suggest that the decreased prolactin levels in treated birds offset their timeline of breeding, so that birds displayed behavior typical of early incubation.