The Pine Island NWR is located on the southwest coast of Florida, north of Sanibel Island in the Pine Island Sound. It is administered as part of the J.N. "Ding" Darling NWR. The refuge was established by President Theodore Roosevelt through Executive Order 939 in 1908 to protect the thousands of herons, egrets, and pelicans that were being hunted to support the plume trade in the early 1900's. The refuge has also been designated as a Florida State Aquatic Preserve.

The 500-acre refuge has been expanded to over 17 islands and consists of densely forested red and black mangroves with little uplands habitat. Indian shell mounds located on Benedict Island show evidence of Calusa Indians once inhabiting the area at the time of European exploration. Several of the islands, including Hemp Island and Bird Key, are important nesting and roosting areas for colonial birds, particularly the Brown pelican. Raccoons are the primary mammal found on the islands and dolphins and manatees can be seen in the surrounding waters.

Small colonies of gopher tortoises may be found on some of the larger islands. Endangered and threatened species include bald eagles, wood storks, sea turtles and manatees.