Mississippi kites (Ictinia mississippiensis) have become an abundant raptor in many urban and exurban areas throughout the Southern Great Plains of the United States. Unfortunately, human–wildlife conflicts have resulted from this juxtaposition of suitable breeding areas for kites and areas that humans frequent, with some kites responding aggressively to humans near nests. To date, there are no data describing the prevalence of aggressive nest defense in the species, making informed management of human and kite conflicts difficult. We assessed and compared the prevalence of aggressive nest-defense by Mississippi kites in an urban area and an exurban area by simulating nest disturbance with a trial pedestrian. Additionally, we examine the relationships between physical features of the nest tree where aggressive behaviors were and were not recorded. Individual kites breeding in the exurban area responded to the trial pedestrian by taking flight from the nesting area, circling overhead, swooping at the pedestrian, or remaining on the nest. In the urban area, kites displayed a more limited suit of responses and either remained on the nest or swooped at the pedestrian. Additionally, kites breeding in the exurban area appeared to respond to experimental disturbance at a greater distance than did urban breeding kites, but not with more attacks on pedestrians. Physical characteristics of the nest tree did not explain aggressive behaviors, thereby suggesting that aggression in Mississippi kites is caused by factors other than nesting location features.