Deer–vehicle collisions (DVCs) are the primary source of mortality for the endangered Florida Key deer (Odocoileus virginianus clavium). Of these collisions, >50% occur on United States Highway 1 (US 1), the primary roadway connecting the islands in the Florida Keys, USA. The DVCs on the 5.6-km section of US 1 on Big Pine Key are responsible for approximately 26% of annual Key deer mortality, but extensive urban development along this highway segment complicates efforts to reduce DVCs using traditional methods (e.g., fencing, underpasses). In 2002, a continuous 2.6-km system of 2.4-m fencing, 2 underpasses, and 4 experimental deer guards was completed. We evaluated the long-term effectiveness of these highway improvements in reducing DVCs within an urban landscape. Deer used the underpasses all 7 postconstruction years (2003–2009) with dramatic increases in use over the course of the project. Roadway fencing largely eliminated deer intrusion onto the fenced area of US 1. Although well-maintained fencing effectively restricted deer movement onto the fenced section of US 1 (73–100% decline), other sections of US 1 experienced increases in DVCs. Overall, highway improvements along the US 1 corridor were effective in reducing Key deer mortality over the long-term. The success of deer guards in preventing deer access into the fenced section of US 1 supported the feasibility of implementing similar wildlife exclusion projects in other urban areas. © 2011 The Wildlife Society.