An array of Great Plains mammals is recorded in the Niobrara Valley. Most species thrive unmanaged, though larger species such as bison and elk occur in fenced enclosures on the Fort Niobrara National Wildlife Refuge and The Nature Conservancy's Niobrara Valley Preserve. A free-ranging moose has also been seen on the Fort Niobrara National Wildlife Refuge.
Federally endangered species like the black-footed ferret once inhabited the area, but have since been extirpated from the region. River otter, a state threatened species, is native to the Niobrara. A reintroduction program was conducted by Nebraska Game and Parks Commission biologists from 1986 to 1992, with a release site near the Sheridan-Cherry County line in northwestern Nebraska. Since then river otter sightings have occurred throughout the Niobrara Valley, including several observations in the 76-mile scenic river reach.