We performed a study to investigate the profile of donor lymphocyte infusion (DLI)-associated acute graft-versus-host disease (GVHD) in haploidentical T-cell-replete hematopoietic stem cell transplantation (HSCT). A total of 124 patients receiving modified DLI after haploidentical T-cell-replete HSCT were enrolled. The cumulative incidence of DLI-associated acute GVHD was 53.2% for grades II–IV and 28.4% for grades III–IV. The duration of GVHD prophylaxis after DLI was the only risk factor for DLI-associated grades III–IV acute GVHD (p < 0.05). The cumulative incidence of grades III–IV acute GVHD in patients with prophylaxis more than six, four to six, two to four, and <2 wk were 9.3%, 14.4%, 31.6%, and 49.5%, respectively (p = 0.018). Furthermore, DLI-associated grades III–IV acute GVHD was the only risk factor for overall survival (p = 0.038, OR = 2.869) and transplant-related mortality (p = 0.018, OR = 3.296) but not a risk factor for relapse after DLI (p = 0.840). This study confirms for the first time that the duration of GVHD prophylaxis after DLI is the only risk factor for the development of grades III–IV acute GVHD. Donor lymphocyte infusion with prophylaxis more than six wk was associated with a lower incidence of grades III–IV acute GVHD.