The Vulnerable Antillean manatee Trichechus manatus manatus (Mammalia, Sirenia) is threatened by hunting, entanglement in fishing nets and habitat degradation but most knowledge of the species comes from coastal and estuarine populations. We therefore studied an Antillean manatee population located >1,100 km from the Caribbean coast, in the Orinoco River of Colombia, during 2001–2005. To verify the occurrence of the manatee and to investigate its habitat use throughout the annual hydrological cycle, we carried out surveys for manatees and manatee feeding signs over c. 40 km of the Orinoco River, between El Burro and Bachaco. We made 870 sightings in 1,003 hours of observation, and found 69 feeding areas in 776 hours of surveys. From 82 interviews in a total of 28 locations in Venezuela and Colombia we compiled 90 reports of manatees injured or killed in the Orinoco during 1980–2004. Both habitat use by the manatee and anthropogenic impacts on the species vary with the annual hydrological cycle. Reproduction appears to occur during the low water period in restricted areas that retain deep water. Improved knowledge of the dynamics of manatee migration and regulation of human activities, especially those related to use of nets and boat traffic, are required for conservation of the Antillean manatee in this freshwater habitat.