Here we present a bioarchaeological analysis of a Postclassic (ca. A.D. 950-1524) Maya mass grave from the site of Zacpetén in northern Guatemala. Osteological and spatial analyses (including a Ripley's K function) found evidence of cutting, drilling, and grinding of long bones and teeth as well as the intentional removal and manipulation of skeletal elements based on the left or right sides of the body. The remains were enveloped in layers of cut blocks and fist-sized chunks of white limestone and were placed in a depression on the western side of the ceremonial core of the site. The western orientation of the depression was explicitly associated with the underworld in contrast with the temple on the eastern side of the ceremonial core. The grave was the product of exhumation and violation of enemies' bodies, sacrifice, or the burial of war dead (or some combination thereof) and was created when the Kowoj group emerged as a political force in the Petén lakes region. It served to symbolically rupture the past inhabitants' links to the site and to create an enduring symbol of their defeat.