Graft versus Host Disease (GvHD) is a major clinical problem with a significant unmet medical need. We examined the role of Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) in a xenogenic GvHD (xeno-GvHD) model induced by injection of human peripheral mononuclear cells (hPBMC) into irradiated NSG mice. Targeting CTLA4 pathway by treatment with CTLA-4 Ig prevented xeno-GvHD, while anti-CTLA-4 antibody treatment exacerbated the lethality and morbidity associated with the GvHD. The xeno-GvHD is associated with infiltration of hPBMCs into the lungs, spleen, stomach, liver, and colon, and an increase in human pro-inflammatory cytokines including IFN-γ, TNFα and IL-5. Infiltration of donor cells and increases in cytokines were attenuated by treatment with CTLA-4 Ig but remained either unaffected or enhanced by anti-CTLA-4 antibody. Further, splenic human T cell phenotyping showed that CTLA-4 Ig treatment prevented the engraftment of human CD45+ cells while anti-CTLA-4 antibody enhanced donor T cell expansion, particularly CD4+ (CD45RO+) subsets including, Tbet+ CXCR3+ and CD25+Foxp3 cells. Comprehensive analysis of transcriptional profiling of human cells isolated from mouse spleen identified a set of 417 differentially expressed genes (DEGs) by CTLA-4 Ig treatment and 13 DEGs by anti-CTLA4 antibody treatment. The CTLA-4 Ig regulated DEGs mapped to down regulated apoptosis, inflammasome, Th17 and Treg pathways, and enhanced TLR receptor signaling, TNF family signaling, complement system, and epigenetic and transcriptional regulation, where as anti-CTLA-4 antibody produced minimal to no impact on these gene pathways. Our results show an important role of co-inhibitory CTLA-4 signaling in xeno-GvHD and suggest therapeutic utility of other immune checkpoint co-inhibitory pathways in the treatment of immune-mediated diseases driven by hyperactive T-cells.