The Antillean manatee Trichechus manatus manatus is categorized as Endangered on the IUCN Red List but little is known about the status of the species in Cuba. Marine protected areas can contribute to manatee conservation in Cuba but the effectiveness of these areas may be jeopardized by a lack of information regarding appropriate design and management. We developed an index of manatee occurrence in the Fauna Refuge Ciénaga de Lanier, Isla de la Juventud, to assess patterns of manatee use in the reserve. We completed 26 field trips during November 2007–October 2013, with a total of 147 survey days. Manatee presence was detected on 47% of survey days and in 96% of field trips; 133 individuals were recorded in 93 sightings. The index of manatee occurrence varied between trips, suggesting a discontinuous use of the area. The mean group size was 1.4 individuals (range 1–5), and calves were observed in 13% of sightings. Observations of manatee behaviour, occurrence and habitat characteristics indicate the importance of the study area as a resting place, refuge and source of fresh water. We recommend that manatee protection be strengthened to avoid human-related mortality and to ensure that the habitats that provide critical resources are given special consideration in future management plans.