Approximately 26% of annual mortality for the endangered Florida Key deer (Odocoileus virginianus clavium) occurs as deer–vehicle collisions (DVCs) on the 5.6-km section of United States Highway 1 (US 1) on Big Pine Key (BPK), but extensive urban development adjacent to sections of US 1 complicates efforts to reduce DVCs. Our objective was to evaluate the effectiveness of the US 1 Project (continuous 2.6-km system of 2.4-m fencing, 2 underpasses, and 4 experimental deer guards constructed on US 1 on BPK) in reducing DVCs along US 1. Deer used the underpasses all 3 postproject years (2003–2005); however, we observed higher underpass use in 2004 and 2005 compared to 2003. Exclusion fencing reduced deer intrusions onto the fenced section of US 1 during the 3-year period (2003, n = 7 deer; 2004, n = 4; 2005, n = 12). With a reduction of deer intrusions onto this section of US 1, DVCs decreased in the fenced area by 73–100%; however, US 1 DVCs within the unfenced sections of US 1 also increased (40%) as expected. In controlling for effects of increasing deer density and traffic volume, study results suggest that highway improvements have decreased the net risk of DVCs along US 1, which indicates that use of deer fencing, deer guards, and underpasses is applicable in other urban communities experiencing unacceptable levels of DVCs.