Abstract Background Though accumulated evidence has demonstrated visceral organ involvement in acute graft-versus-host disease (aGVHD), how aGVHD influences the bone marrow (BM) niche and the reconstitution of hematopoiesis post-hematopoietic stem cell transplantation remains largely unknown. Methods In the current study, the cell morphology, immunophenotype, multi-differentiation capacity, self-renewal capacity, and hematopoiesis promotion of the MSCs from aGVHD and non-aGVHD patients were investigated. Additionally, the stemness and hematopoiesis-promoting property of healthy donor-derived MSCs were evaluated in the presence of BM supernatant from aGVHD patients. Mechanistically, antibodies targeting inflammatory cytokines involved in aGVHD were added into the MSC culture. Furthermore, a recombinant human tumor necrosis factor (TNF-α) receptor-Ig fusion protein (rhTNFR:Fc) was used to protect healthy donor-derived MSCs. Moreover, mRNA sequencing was performed to explore the underlying mechanisms. Results The aGVHD MSCs exhibited morphological and immunophenotypic characteristics that were similar to those of the non-aGVHD MSCs. However, the osteogenic and adipogenic activities of the aGVHD MSCs significantly decreased. Additionally, the colony formation capacity and the expression of self-renewal-related genes remarkably decreased in aGVHD MSCs. Further, the hematopoiesis-supporting capacity of aGVHD MSCs significantly reduced. The antibody neutralization results showed that TNF-α contributed to the impairment of MSC properties. Moreover, rhTNFR:Fc exhibited notable protective effects on MSCs in the aGVHD BM supernatants. The mRNA sequencing results indicated that the TNF-α pathway and the Toll-like receptor pathway may be activated by TNF-α. Conclusions Thus, our data demonstrate MSCs as cellular targets of aGVHD and suggest a potential role of TNF-α blockage in maintaining the BM niche of aGVHD patients.