Corridors may mitigate the adverse effects of habitat fragmentation by restoring or maintaining connectivity between disjunct populations. The efficacy of corridors for large carnivores, however has rarely been evaluated objectively. We used noninvasive sampling, microsatellite analysis, and population assignment tests to evaluate the effectiveness of a regional corridor in connecting two Florida black bear (Ursus americanus floridanus) populations (Osceola and Ocala). Bear movement was predominantly unidirectional, with a limited mixing of individuals from the two populations in one area of the corridor We also documented bears in Osceola that were genetically assigned to Ocala and bears in Osceola that may be offspring from an Osceola-Ocala mating. Our results indicate that the Osceola-Ocala corridor is functional and provides a conduit for gene flow between these populations. Human development, however may hinder the use of the Osceola-Ocala corridor by bears. The noninvasive sampling and genetic methods we used provide a means of evaluating corridor effectiveness that can help identify linkages necessary for maintaining metapopulation structure and population viability.