The giant otter (Pteronura brasiliensis) is an endangered mammal that occupies aquatic environments, especially in the Amazon Rainforest and Pantanal wetlands in South America. The species uses shelters (dens and campsites), such as burrowed tunnels and tangles of branches and roots along watercourses, where it feeds mainly on fishes. In Espírito Santo Creek (northern portion of the Pantanal wetlands in the state of Mato Grosso, Brazil), 60 shelters (45 dens and 15 campsites) were identified and georeferenced during 12 fieldtrips between 2002 and 2003. The density was 1.8 dens/km. The shelters were significantly found in locations with partial (35%) or complete (55%) plant cover. The dens were dug mainly in soil associated with roots located, on average, 4.4 m in distance and 2.4 m in height in relation to the waterline. The campsites had a significantly smaller distance and height compared to the dens. The greater density of dens in the study area underscores the need for conserved environments with little human interference for the protection of giant otter populations. The vegetal coverage of riparian forests and the occurrence of ravines along Espírito Santo Creek offer greater protection to the shelters, hindering the access of predators to the interior of the shelter. Studies integrating biological and ecological knowledge with social participation in areas of occurrence of the giant otter are fundamental to the conservation of the species and its habitat in the Pantanal wetlands of Mato Grosso, Brazil.