We investigated Key deer mortality data between 1966 and 2017 and found that about 75% of all reported Key deer mortalities were related to collisions with vehicles. We investigated where the greatest concentrations of Key deer-vehicle collisions were after the eastern section of US Hwy 1 on Big Pine Key was mitigated with a wildlife fence, 2 underpasses, and 4 deer guards. We also investigated the effectiveness of these mitigation measures in reducing Key deer-vehicle collisions. The wildlife fence and associated mitigation measures along the eastern section of US Hwy 1 on Big Pine Key were highly effective (81.3-93.9%) in reducing Key deer-vehicle collisions along that road section. However, there was an increase in collisions in the unmitigated section of US Hwy 1, both in absolute numbers and expressed as a percentage of the total Key deer population size. Thus, the overall Key deer road mortality on US Hwy 1 was not reduced but it was moved from the mitigated section to the unmitigated section of US Hwy 1. After mitigation, a significant hotspot of Key deer-vehicle collisions appeared at the western fence end indicating a "fence-end run". Additional significant Key deer-vehicle collision hotspots occurred further west along the unmitigated highway section on Big Pine Key. The results illustrate the need to implement measures at the appropriate spatial scale. In order to reduce the overall number of Key deer-vehicle collisions along US Hwy 1, the entire section of US Hwy 1 on Big Pine Key would need to be mitigated. Ideally, the wildlife fence should be extend to the western side of Big Pine Key with additional safe crossing opportunities for Key deer and other wildlife species. However, the western section of US Hwy 1 that is currently unfenced has many buildings and access roads for business and residences. Rather than recommending a particular mitigation option for this road section, we summarized the pros and cons of eight different strategies aimed at reducing Key deer-vehicle collisions along the developed section of US Hwy 1, including a "No Action" option.