Purpose: Currently, data on the role of tacrolimus and minidose methotrexate (MTX) in pediatric unrelated hematopoietic stem cell transplantation (HSCT) is limited. We report the outcomes of unrelated hematopoietic stem cell recipients, evaluating engraftment status, incidence of acute and chronic graft versus host disease (GVHD) and toxicities after use of tacrolimus and minidose MTX for GVHD prophylaxis. Method: From January 2004 to December 2013, 35 children who received tacrolimus and minidose MTX as prophylaxis were reviewed. All patients received tacrolimus beginning the day prior to transplant at a dose of 0.03 mg/kg/day by continuous intravenous (IV) infusion. Tacrolimus levels were monitored daily and dosages adjusted to maintain serum levels 5-15 ng/mL. MTX was administered at a dose of 5 mg/m2 IV on days 1, 3, 6 and 11. Results: Median age at transplantation was 8.4 years (range 0.75–18.9 ). 17 patient received human leukocyte antigen fully matched donor transplants and 18 received partially mismatched transplants. Stem cell sources included 25 peripheral blood donors, 4 bone marrow donors and 6 umbilical cord blood donors. Diagnoses included 11 acute lymphoblastic leukemias, 10 acute myeloid leukemias, 6 Fanconi anemias and 5 severe aplastic anemias. All patients except 2, who had unrelated cord blood transplants, were successfully engrafted. The median time to ANC recovery (ANC＞500x10 6/ul) was 12 days. The incidence of acute GVHD was 33.3% including 15.1% of grade III-IV GVHD. Localized chronic GVHD developed in only 2 of 27 (7.4%) evaluable patients. Lower tacrolimus levels in day 1-21 were associated with a higher incidence of acute GVHD (p=0.033). Other adverse events included positive CMV antigenemia (37.1%), hemorrhagic cystitis (28.6%), veno-occlusive disease of the liver (22.8%), and post-transplant lymphoproliferative disorder (5.7%). The estimated 4-year event free survival and overall survival of the patients were 71.2% and 80.0%, respectively. Common adverse effects of tacrolimus included hypomagnesemia (60.0%), nephrotoxicity (11.4%), hyperglycemia (11.4%), hypertension (11.4%). Conclusion: Overall, the combination of tacrolimus and minidose MTX could be effectively administered in the setting of pediatric unrelated SCT. In addition, tacolimus level monitoring after unrelated SCT is important in preventing acute GVHD.