Introduction: This prospective study evaluated a calcineurin inhibitor-free graft-versus-host disease (GVHD) prophylaxis regimen of ruxolitinib in combination with post-transplant cyclophosphamide (PTCy). Patents and Methods: Twenty patients with primary or secondary myelofibrosis were prospectively enrolled. Reduced intensity conditioning was performed, followed by allogeneic stem cell transplantation from related (n = 7) or unrelated (n = 13) donors. GVHD prophylaxis included only PTCy and ruxolitinib (45 mg) from day–7 to day–2, and 15 mg from day+5 to day+100. This trial was registered at www.clinicaltrials.gov as #NCT02806375. Results: Primary engraftment was documented in 17 patients. One patient experienced primary graft failure and 2 died before engraftment. Eleven patients demonstrated severe poor graft function (SPGF), which required ruxolitinib dose reduction. The regimen was well tolerated, with grade 3–4 non-haematological toxicity in 30%, viral reactivation in 45%, and severe sepsis in 15% of patients. The incidence of acute GVHD grade II–IV was 25%, grade III-IV GVHD was 15%, and moderate chronic GVHD was 20%, with no severe cases. Only 2 patients required systemic steroids. Haematological relapse was documented in 1 patient. Two-year non-relapse mortality was 15%, 2-year overall survival was 85%, and 2-year event-free survival was 72%. Conclusion: GVHD prophylaxis with PTCy and ruxolitinib is associated with low toxicity, good acute and chronic GVHD control, and low relapse incidence. However, the relatively high rate of SPGF should be taken into account. SPGF could possibly be mitigated by ruxolitinib dose reduction.