Leptospirosis is a zoonotic disease caused by the bacterium Leptospira interrogans. The organism is typically maintained within a geographic region by colonizing renal tubules of carrier animals and shed into the environment in urine. We assessed whether L. interrogans was present in fox squirrels (Sciurus niger) in Larimer County, Colorado, USA, and whether it is associated with disease. Twenty-two squirrels were trapped from 29 November 2011 to 15 December 2011 for use in an unrelated study. The squirrels were individually housed for 33–65 days and euthanized; no clinical disease was observed. On gross examination, significant renal lesions were observed in 6 of 22 animals (27%). Histologically, affected animals had severe neutrophilic tubulitis with interstitial nephritis. Immunohistochemistry was conducted on the kidneys of all animals and 10 of 22 (45%) were positive for L. interrogans, with varying severity of infection. The same 10 squirrels were serologically positive for antibodies specific to L. interrogans. These results suggest that L. interrogans is present in fox squirrels in Larimer County, Colorado, USA, and may be associated with varying degrees of renal disease. Further investigation into the role of wildlife in the ecology of leptospirosis within the region is warranted.