The immune plasticity of the host critically influences disease progression and therapeutic outcomes. In acute graft-versus-host disease (aGVHD), conventional conditioning regimens often induce toxicity, underscoring the need for effective adjunctive strategies to restore immune balance. This study evaluates the immunomodulatory and regenerative potential of secretome derived from naive and preconditioned human mesenchymal stem cells (MSCs) in aGVHD. Human MSCs from bone marrow and Wharton’s Jelly were preconditioned under hypoxia (1% O₂), apoptosis (1 µM staurosporine, 24 h), or both (dual preconditioning), and their secretome was assessed for immunoregulatory and antioxidant effects using T-cell proliferation assays, Treg induction, and M1-to-M2 macrophage polarization. Cellular bioenergetics and mitochondrial function were analyzed to explore metabolic reprogramming. Mechanistic insights were obtained through LC-MS/MS-based proteomic profiling of CCM co-cultured with aGVHD patient-derived activated PBMNCs, and in vivo efficacy was validated in a chemotherapy-induced aGVHD murine model. Dual preconditioned WJ-MSCs (WJ-MSCsHYP+APO)-derived secretome exhibited superior immunomodulatory activity, suppressing T-cell proliferation, enhancing Treg and Th2/Th9 differentiation, and promoting M2 macrophage polarization. It reduced mitochondrial ROS, improved mitochondrial polarization, and shifted T-cell metabolism from glycolysis to oxidative phosphorylation. Proteomic analysis revealed downregulation of IL-12, IL-17, and JAK–STAT signaling, along with modulation of complement, coagulation, and metabolic pathways. Secretome also enhanced extracellular matrix remodeling and antioxidant responses, supporting tissue repair. Dual preconditioning significantly amplifies the immunoregulatory, antioxidant, and regenerative efficacy of WJ-MSCs-derived secretome, presenting a promising non-cellular therapeutic approach for aGVHD management.