Graft-versus-host disease (GVHD) is a major complication following donor stem cell transplantation, where donor T cells react against host tissues. Post-transplant cyclophosphamide (PTCy) targets these reactive T cells to reduce but not eliminate GVHD in humans and humanised mice. Interleukin-6 receptor (IL-6R) blockade can increase regulatory T cells (Tregs) and has been used in preclinical trials for GVHD, with some success. The aim of this study was to determine the effect of PTCy combined with IL-6R blockade on GVHD and immune cell subsets in a humanised mouse model. Immunodeficient NOD-scid-IL2Rγnull (NSG) mice were injected intraperitoneally (i.p.) with 20 x 106 human peripheral blood mononuclear cells (hPBMCs) on Day 0. Mice were injected i.p. with either PTCy (33mg/kg) or saline on Days 3 and 4, and with either tocilizumab (anti-IL-6R) or a control antibody (0.5mg/mouse) on Day 0 then twice weekly for 4 weeks. Mice were monitored for clinical GVHD and human cell engraftment assessed by flow cytometry. Prolonged survival was observed in PTCy + tocilizumab (MST, 48 days) and PTCy (MST, 66 days) compared to tocilizumab (MST, 38 days) and control mice (saline + control antibody) (MST, 26 days), however the addition of tocilizumab did not improve survival compared to PTCy alone (P=0.1967). All groups showed similar engraftment of splenic hCD45+ cells (55-60%), but PTCy + tocilizumab mice demonstrated increased splenic hCD4+hCD25+hCD127lo Tregs (10.6%) compared to PTCy (6.5%) (P=0.0136), tocilizumab (6.9%) (P=0.0297) and control mice (5.6%) (P=0.0012) at 4 weeks post-injection. Serum interferon-gamma concentration was significantly reduced in PTCy + tocilizumab (1.2pg/mL) compared to control (2.3pg/mL) (P=0.0406) mice, but not PTCy alone mice (1.5pg/mL) (P=0.8332). This study demonstrated that combining PTCy and IL-6R blockade increases Tregs and reduces interferon-gamma at 4 weeks, but does not improve survival, providing insight into the effects of this combination therapy on GVHD outcomes.