Cannabidiol (CBD) is a highly lipidic phytocannabinoid with remarkable anti-inflammatory effects. The aim of this study was to evaluate CBD's effects and mechanisms of action in the treatment of mice subjected to acute graft-versus-host disease (aGVHD). aGVHD was induced by the transplantation of bone marrow cells and splenocytes from C57BL-6j to Balb-c mice. The recipient mice were treated daily with CBD, and the treatment reduced mouse mortality by decreasing inflammation and injury and promoting immune regulation in the jejunum, ileum, and liver. Analysis of the jejunum and ileum showed that CBD treatment reduced the levels of C-C motif chemokine ligand (CCL) 2, CCL3, CCL5, tumor necrosis factor , and interferon (IFN). CCL3 and IFN levels were also decreased in the liver. Mechanistically, CBD also increased the number of cannabinoid receptor type 2 (CB) receptors on CD4 and forkhead box P3 cells in the intestine, which may explain the reduction in proinflammatory cytokines and chemokines. Antagonists of the CB receptor reduced the survival rates of CBD-treated mice, suggesting the participation of this receptor in the effects of CBD. Furthermore, treatment with CBD did not interfere with the graft-versus-leukemia response. CBD treatment appears to protect aGVHD mice by anti-inflammatory and immunomodulatory effects partially mediated by CB receptor interaction. Altogether, our study suggests that CBD represents an interesting approach in the treatment of aGVHD, with potential therapeutic applications in patients undergoing bone marrow transplantation. SIGNIFICANCE STATEMENT: This study provides for the first time a mechanism by which cannabidiol, a phytocannabinoid with no psychoactive effect, induces immunomodulation in the graft-versus-host disease. Enhancing intestinal cannabinoid receptor type 2 (CB) receptor expression on CD4 and forkhead box P3 cells and increasing the number of these regulatory cells, cannabidiol decreases proinflammatory cytokines and increases graft-versus-host disease mice survival. This effect is dependent of CB receptor activation. Besides, cannabidiol did not interfere with graft-versus-leukemia response, a central response to avoid primary disease relapse.