Humboldt Penguins (Spheniscus humboldti) are globally Vulnerable due to the decline of the population and to a decline in the number of breeding sites in comparison to historical records. In 2010, we observed a few thousand Humboldt Penguins nesting in a surface-nest colony on Isla Santa Rosa in southern Perú. This number was unexpected because this site has never been listed as an important Humboldt Penguin breeding ground. We visited this island again in June 2011 and 2012, and counted 1,965 and 1,745 active nests, respectively. These numbers indicate not only the presence of at least 3,500–4,000 Humboldt Penguins, but places Isla Santa Rosa among the five largest Humboldt Penguin colonies within its entire range. The need for recognition of Isla Santa Rosa as a major Humboldt Penguin refuge is crucial for the conservation of the species, as the island has remained untouched from guano harvesting since 1996. This has resulted in a significant accumulation of guano likely to be legally collected in the next few years. Unsupervised guano harvesting could cause a Humboldt Penguin exodus and jeopardize the existence of this colony. Under this scenario, it is recommended that Isla Santa Rosa be closed to guano harvesting until a surveillance program to protect the Humboldt Penguins can be established and proven to work under the conditions found on Isla Santa Rosa.