Cyclosporine (CsA) is widely used for graft-versus-host disease (GVHD) in pediatric hematopoietic stem cell transplantation (HSCT); however, the optimal schedule of its administration has not been established. We retrospectively compared the time-course of blood CsA levels and the incidence of CsA-associated adverse effects, grade II-IV acute GVHD, and chronic GVHD among 26 pediatric HSCT recipients who were receiving CsA by continuous infusion (CIF) (n=8) or by 3h infusion twice-daily (3TD) (n=18). In the 3TD group, the target level of the C3 value, which is strongly co-related with the area under the concentration-time curve, was maintained without any serious adverse effects in most patients. No significant differences were observed in the incidence of grade II-IV acute GVHD and chronic GVHD between CIF and 3TD groups. 3TD in combination with C3 monitoring enables safe and easy control of CsA blood levels and is thought to be useful for GVHD prophylaxis in pediatric HSCT.