Graft-versus-host disease (GVHD) is a T cell-mediated inflammatory disorder that arises following donor blood stem cell transplantation and is associated with a loss in donor regulatory T cells (Tregs). P2X7, an extracellular ATP-gated cation channel, functions as a damage-associated molecular pattern receptor on immune cells and plays important roles in inflammation, with emerging roles in GVHD. This study examined whether an anti-human P2X7 monoclonal antibody (mAb) (clone L4) could impact disease progression in a humanised mouse model of GVHD. Immunolabelling and flow cytometry confirmed that this mAb could bind human but not murine P2X7. Similarly, flow cytometric measurements of ATP-induced cation uptake confirmed that this mAb could block human but not murine P2X7 activity. Humanised mice, established by injecting NSG mice with human peripheral blood mononuclear cells (10 million cells i.p. per mouse, Day 0), were injected every second day (Days 0-8) with the anti-P2X7 or control mAb (100 μg i.p. per mouse), and examined for clinical GVHD for up to 75 days. Human cells in tissues were examined at Day 21 (pre-GVHD onset) and humane endpoint (late GVHD) by immunolabelling and flow cytometry. The anti-P2X7 mAb did not impair human leukocyte or effector T cell engraftment at either time point. Clinical GVHD in anti-P2X7 mAb treated mice was significantly reduced over time (endpoint) compared to control mice. Anti-P2X7 mAb treatment significantly increased proportions of human Tregs in the spleens (Day 21 and endpoint) and livers (endpoint) of mice compared to control mice. These studies indicate that blockade of P2X7 with the anti-human P2X7 mAb reduces GVHD progression in humanised mice and this corresponds with an increase in human Tregs. These studies provide the first direct evidence for a role for donor (human) P2X7 in GVHD progression.