We assessed the potential of white-tailed deer (WTD) (Odocoileus virginianus) to be a sentinel for human cases of Lyme disease (LD) in Indiana using location data from a 3-year survey of approximately 3400 hunted deer with associated tick Ixodes scapularis and Borrelia burgdorferi (Bb) data. Data on human LD cases at the county level were obtained from the Indiana Department of Health. All data were assigned to county centroids to match the resolution of the LD data before creating optimized trend surfaces for LD incidence, hunted deer count, Ixodes scapularis and Bb prevalence. To determine whether LD was spatially associated with the areas of high densities of deer, deer with Ixodes scapularis and deer with ticks infected with Bb, we used spatial analysis with distance indices (SADIE). The SADIE analysis found significant spatial association between LD and the distribution of three organismal predictor variables, that is, WTD, Ixodes ticks and Bb. Lyme disease incident rate varied between 0.08 cases per 10 000 habitants (Johnson county) and 5.9 cases per 10 000 habitants (Warren county). In conclusion, WTD can be used as an accurate and cost-effective sentinel for human LD. This method will permit public health workers to identify potentially endemic areas independently of human case reports.