Background and Aims: The safety and efficacy of mes-enchymal stem cells (MSCs) in the treatment of acute-on-chronic liver failure (ACLF) have been validated. However, the impact of the pathological ACLF microenvironment on MSCs is less well understood. This study was designed to explore the changes in the functional properties of MSCs exposed to ACLF serum. Methods: MSCs were cultured in the presence of 10％, 30％ and 50％ serum concentra-tions from ACLF patients and healthy volunteers. Then, the cell morphology, phenotype, apoptosis and prolifera-tion of MSCs were evaluated, including the immunosup-pressive effects. Subsequently, mRNA sequencing analysis was used to identify the molecules and pathways involved in MSC functional changes in the context of ACLF. Re-sults: In the presence of ACLF serum, MSC morphology significantly changed but phenotype did not. Besides, MSC proliferation activity was weakened, while the apoptosis rate was lightly increased. Most importantly, the immu-nosuppressive function of MSCs was enhanced in a low-concentration serum environment but transformed into a proinflammatory response in a high-concentration serum environment. RNA sequencing indicated that 10％ serum concentration from ACLF patients mediated the PI3K-Akt pathway to enhance the anti-inflammatory effect of MSCs, while the 50％ serum concentration from ACLF patients promoted the conversion of MSCs into a proinflammato-ry function by affecting the cell cycle. Conclusions: The 50％ ACLF serum concentration is more similar to the en-vironment in the human body, which means that direct peripheral blood intravenous infusion of MSCs may reduce the effect of transplantation. Combining treatments of plasma exchange to reduce harmful substances in serum may promote MSCs to exert a stronger anti-inflammatory effect.