Grooming is a common animal behavior that aids in ectoparasite defense. Ectoparasites can stimulate grooming, and natural selection can also favor endogenous mechanisms that evoke periodic bouts of “programmed” grooming to dislodge or kill ectoparasites before they bite or feed. Moreover, grooming can function as a displacement or communication behavior. We compared the grooming behaviors of adult female black-tailed prairie dogs (Cynomys ludovicianus) on colonies with or without flea control via pulicide dust. Roughly 91% of the prairie dogs sampled on the non-dusted colony carried at least one flea, whereas we did not find fleas on two dusted colonies. During focal observations, prairie dogs on the non-dusted colony groomed at higher frequencies and for longer durations than prairie dogs on the dusted colonies, lending support to the hypothesis that fleas stimulated grooming. However, the reduced amount of time spent grooming on the dusted colonies suggested that approximately 25% of grooming might be attributed to factors other than direct stimulation from ectoparasites. Non-dusted colony prairie dogs rarely autogroomed when near each other. Dusted colony prairie dogs autogroomed for shorter durations when far from a burrow opening (refuge), suggesting a trade-off between self-grooming and antipredator defense. Allogrooming was detected only on the non-dusted colony and was limited to adult females grooming young pups. Grooming appears to serve an antiparasitic function in C. ludovicianus. Antiparasitic grooming might aid in defense against fleas that transmit the plague bacterium Yersinia pestis. Plague was introduced to North America ca. 1900 and now has a strong influence on most prairie dog populations, suggesting a magnified effect of grooming on prairie dog fitness.