Microglia, the resident immune cells of the central nervous system, exist in either a “resting” state associated with physiological tissue surveillance or an “activated” state in neuroinflammation. We recently showed that ATP is the primary chemoattractor to tissue damage in vivo and elicits opposite effects on the motility of activated microglia in vitro through activation of adenosine A_2A receptors. However, whether systemic inflammation affects microglial responses to tissue damage in vivo remains largely unknown. Using in vivo two‐photon imaging of mice, we show that injection of lipopolysaccharide (LPS) at levels that can produce both clear neuroinflammation and some features of sepsis significantly reduced the rate of microglial response to laser‐induced ablation injury in vivo. Under proinflammatory conditions, microglial processes initially retracted from the ablation site, but subsequently moved toward and engulfed the damaged area. Analyzing the process dynamics in 3D cultures of primary microglia indicated that only A_2A, but not A₁ or A₃ receptors, mediate process retraction in LPS‐activated microglia. The A_2A receptor antagonists caffeine and preladenant reduced adenosine‐mediated process retraction in activated microglia in vitro. Finally, administration of preladenant before induction of laser ablation in vivo accelerated the microglial response to injury following systemic inflammation. The regulation of rapid microglial responses to sites of injury by A_2A receptors could have implications for their ability to respond to the neuronal death occurring under conditions of neuroinflammation in neurodegenerative disorders. GLIA 2014;62:1345–1360