For relapsed acute myeloid leukemia (AML) patients who received allogeneic hematopoietic stem cell transplantation, donor lymphocyte infusion (DLI) is an effective therapy. However, the cell source of DLI remains a topic of debate. In this study, we aimed to compare the efficacy and safety of G-CSF mobilized cells (G-DLI) with conventionally collected DLI (C-DLI). A total of 81 patients (50 C-DLI vs. 31 G-DLI) were assessed for clinical outcomes. There were no statistically significant differences in the baseline characteristics between the two groups including AML risk, donor types, interval from relapse to DLI, and infused CD3⁺ cell count. Although not statistically significant, complete remission (CR) and chimerism conversion rates were higher in G-DLI than in C-DLI: 51.6% vs. 28.0%, P = 0.057 and 42.3% vs. 28.2%, P = 0.363, respectively. There was no difference in acute graft-versus-host disease (GVHD) incidence and severity of acute GVHD between the two groups. The median overall survival (OS) of the G-DLI and C-DLI groups was 139 days and 106 days, respectively (P = 0.58). In conclusion, G-DLI appears to be a safe and an equally efficacious substitute for C-DLI, which is more readily available.