This study addressed behavioral responses by black-tailed prairie dogs (Cynomys ludovicianus) to human intrusion in urban and rural environments in Boulder, Colorado. We expected that if prairie dogs habituate to repeated disturbances, they should allow a recurring human intruder to approach closer over time before sounding an alarm bark or initiating concealment. We also predicted that urban colonies could be approached more closely than rural colonies before displaying an avoidance response. Four colonies (2 rural and 2 urban) were approached >100 times over a 7-month period. Rather than exhibiting habituation, prairie dogs demonstrated increased responsiveness in concealment behavior, retreating to their burrows earlier, with recurring disturbances. Barking distances did not change consistently with repeated intrusion, but, over time, prairie dogs barked less frequently when performing their avoidance response, a result with implications for prairie dog management. Rural colonies had higher initial concealment distances, and these distances increased more rapidly with repeated intrusion than did concealment distances in urban colonies. Thus, rural prairie dogs may be more sensitive to human intrusion than urban prairie dogs.