The widespread encroachment of woody plants throughout the semi-arid grasslands in North America has largely resulted from overgrazing by domestic livestock, fire suppression, and loss of native large and small mammalian herbivores. Burrowing-herbivorous mammals, such as prairie dogs (Cynomys spp.), help control shrub encroachment through clipping of shrubs and consumption of their seedlings, but little is known about how this important ecological role interacts with and may be influenced by co-existing large herbivores, especially domestic livestock. Here, we established a long-term manipulative experiment using a 2 × 2 factorial design to assess the independent and interactive effects of black-tailed prairie dogs (Cynomys ludovicianus) and cattle (Bos taurus) on honey mesquite (Prosopis glandulosa) abundance and structure. We found that, after five years, mesquite abundance was three to five times greater in plots where prairie dogs were removed compared to plots where they occurred together or alone, respectively. While both prairie dogs and cattle reduced mesquite cover, the effect of prairie dogs on reducing mesquite abundance, cover, and height was significantly greater than that by cattle. Surprisingly, cattle grazing enhanced prairie dog abundance, which, in turn, magnified the effects of prairie dogs on mesquite shrubs. Mesquite canopy cover per hectare was three to five times greater where prairie dogs and cattle were absent compared to where they occurred together or by themselves; whereas, cumulative mesquite height was two times lower on sites where prairie dog and cattle occurred together compared to where they occurred alone or where neither occurred. Data from our experimental study demonstrate that prairie dogs and moderate grazing by cattle can suppress mesquite growth, and, when their populations are properly managed, they may interact synergistically to significantly limit mesquite encroachment in desert grasslands.