Key Cave NWR, about 5 miles southwest of Florence, Alabama, was established in 1997 to ensure the biological integrity of Key Cave, Collier Cave, and the aquifer common to both. Key Cave has been designated as critical habitat for the endangered Alabama cavefish (Speoplatyrhinus poulsoni) and as a priority one maternity cave for the endangered gray bat (Myotis grisescens). Collier Cave, located approximately 1.5 miles upstream from Key Cave and within the acquisition boundary, is important to both species as potential habitat. Both caves are on the northern shore of Pickwick Lake in a limestone karst area that contains numerous sinkholes and several underground cave systems. The area's sinkholes are an integral component of groundwater recharge to the caves. The area directly north of Key Cave was identified as a potential high hazard risk area for groundwater recharge and this is where the 1,060-acre Refuge was established.

The Refuge is the only known location of the Alabama cavefish which inhabits the underground pools in Key Cave. Based on the apparent distribution, the number of specimens collected, and the number of individuals observed, this small fish appears to be the rarest of American cavefish and one of the rarest of all freshwater fish. Two species of blind crayfish (Procambarus pecki and Cambarus jonesi) also inhabit Key Cave. Key Cave is also a priority one maternity cave for the endangered gray bat. Gray bat emergence counts are conducted annually and have averaged 33,400 gray bats since inception of the refuge in 1997. Approximately 5,000 young gray bats are produced annually by this maternity colony. The Refuge is currently 1,060 acres, but may encompass 1,800 acres upon completion of additional purchases within the Key Cave recharge area.

In addition to the endangered species mentioned above, the refuge provides habitat for a variety of migratory and resident wildlife species. One hundred and sixty bird species have been sighted on the refuge. Several bird species commonly seen during the breeding season include dickcissels, grasshopper sparrows, field sparrows, and northern bobwhite, which are indicative of the native grasslands and early successional habitats found on the refuge. Northern harriers can be seen flying low over refuge grasslands searching for prey during the winter months. Short-eared owls can also be seen occasionally in grasslands during the winter. Other commonly seen wildlife species include cottontail rabbits, coyotes, white-tailed deer, gray squirrels, eastern meadowlarks, horned larks, and eastern bluebirds.

Lands within the Key Cave recharge area have been in agricultural production for many years. Historically, lands that are now part of the refuge were used primarily for cotton farming. Key Cemetery, which may have been a slave cemetery, is located on the refuge and may yield clues as to the historical land use of the refuge.

Key Cave lies on the northern shore of Pickwick Lake in a limestone karst area that contains numerous sinkholes and several underground cave systems. The areas sinkholes are an integral component of groundwater recharge for the cave. In 1990, the Ozark Underground Laboratory determined the underground recharge area by the use of dye traces. The recharge area was divided into four potential risk areas: high hazard, moderately high hazard, moderate hazard, and low hazard.

The Monsanto Company owned a large 1,060 acre tract in the high hazard risk area that was identified by conservationists as in need of protection and in 1992 sold this tract to The Conservation Fund. The Conservation Fund held the land until the U.S. Fish and Wildlife Service bought the land five years later to establish Key Cave NWR, which became part of the National Wildlife Refuge System on January 3, 1997.