Non-native black-tailed prairie dogs (Cynomys ludovicianus Ord, 1815) that had emigrated from a private colony were reported by property owners in rural North Central Florida in 2006. The Florida Fish and Wildlife Conservation Commission and U.S. Department of Agriculture Animal and Plant Health Inspection Service Wildlife Services (WS) initiated an assessment of the population to determine its current distribution and the feasibility of eradication. Multiple surveys, interviews with residents and observations of abandoned burrows confirmed movement of animals as far as 3 km from the source colony. WS personnel conducted an eradication program of the source colony from 23 March through 4 May 2009, primarily using a 0.17-caliber rifle along with cable restraint devices for particularly wary animals. A total of 85 prairie dogs were killed at the site of the source colony and one animal was killed on a separate property. Subsequent surveys from 2009 through 2012 confirmed the eradication was successful. Communication with the owner of the source colony and nearby residents throughout the project was critical to success.