Lontra canadensis (North American River Otter; hereinafter “River Otter”) is a widespread, semi-aquatic mustelid. Despite considerable research and management of the species, there is limited information on the initiation of natal denning and characteristics of natal den habitat, especially in the southeastern United States. In February 2020, during a survey for bats in a cave in Jackson County, FL, we encountered a River Otter natal den containing 3 pups and an adult. The den was located in a small side chamber within the cave, 9.6 m from the nearest entrance and in an area with little available light. The nearest cave entrance was 492 m from the closest permanent body of water. We estimate that the pups were born in late January. Caves may make ideal natal dens because of reduced disturbance by predators, a stable temperature and humidity, and protection from harsh weather.