Florida Panther NWR was established in 1989 under the authority of the Endangered Species Act to protect the Florida panther and its habitat. The refuge consists of 26,400 acres and is located within the heart of the Big Cypress Basin in southwest Florida. This subtropical ecosystem covers more than 2,400 square miles of diverse wetland and upland habitat types. The refuge encompasses the northern origin of the Fakahatchie Strand, which is the largest cypress strand in the Big Cypress Swamp. Orchids and other rare plants are found within the refuge. The refuge contains a diverse mix of pine forests, cypress domes and strands, wet prairies, hardwood hammocks, and lakes. Besides the panther, 24 other species of mammals, birds and reptiles found in and around the refuge are state or federally listed as threatened, endangered or of special concern. The Florida black bear, alligator, wood storks, limpkin, swallow-tailed kite, indigo snake, Everglades mink, and Big Cypress fox squirrel are a few examples. Other resident wildlife include white-tailed deer and feral hogs, which are prey for panthers. Wild turkey and bobwhite quail also can be found on the refuge. Florida panthers den, hunt and roam throughout the refuge and adjacent lands. The refuge is located within the core of the heaviest, radio-collared panther distribution. The refuge is utilized by 5-11 collared panthers each month.

The Florida Panther NWR is located 20 miles east of Naples, FL, on the northwest corner of the intersection of I-75 and State Road 29. The headquarters for the refuge is located at 3860 Tollgate Blvd, Naples, FL, within the Comfort Inn at exit 101 on I-75.