Background: The use of unmanipulated haploidentical hematopoietic stem cell transplantations (haplo-HSCT) with post-transplant cyclophosphamide (PTCY) in children has emerged as an acceptable alternative to the patients without a matched donor. However, the timing of calcineurin inhibitors (CNIs) used in combination with PTCY is increasingly becoming a topic of controversy. Method: We evaluated 49 children with acute leukemia who underwent unmanipulated haplo-HSCT with PTCY according to the initiation day of CNIs (pre- or post-cyclophosphamide [CY]). Results: There were no significant differences in the overall survival analysis between the 2 groups. The cumulative incidence of relapse at 2 years was 21.2% in the pre-CY group and 38.9% in the post-CY group (p = 0.33). Cytokine release syndrome (CRS) was observed more frequently in the post-CY group (p = 0.04). The overall survival and event-free survival at 2 years in patients with and without CRS in the pre-CY group were 42.9% versus 87.5% (p = 0.04) and 38.1% versus 87.5% (p = 0.04), respectively. Conclusion: Our study shows that the argument for starting CNI administration after CY is tenuous, and the rationale for not starting CNIs before CY needs to be reconsidered.