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Abstract Genetic rescue—an increase in population fitness following the introduction of new alleles—has been proven to ameliorate inbreeding depression in small, isolated populations, yet is rarely applied as a conservation tool. A lingering question regarding genetic rescue in wildlife conservation is how long beneficial effects persist in admixed populations. Using data collected over 40 years from 1192 endangered Florida panthers (Puma concolor coryi) across nine generations, we show that the experimental genetic rescue implemented in 1995—via the release of eight female pumas from Texas—alleviated morphological, genetic, and demographic correlates of inbreeding depression, subsequently preventing extirpation of the population. We present unequivocal evidence, for the first time in any terrestrial vertebrate, that genetic and phenotypic benefits of genetic rescue remain in this population after five generations of admixture, which helped increase panther abundance (> fivefold) and genetic effective population size (> 20-fold). Additionally, even with extensive admixture, microsatellite allele frequencies in the population continue to support the distinctness of Florida panthers from other North American puma populations, including Texas. Although threats including habitat loss, human-wildlife conflict, and infectious diseases are challenges to many imperiled populations, our results suggest genetic rescue can serve as an effective, multi-generational tool for conservation of small, isolated populations facing extinction from inbreeding.