Background <p>The effect of disease conditions on the differentiation capability of mesenchymal stem cells (MSCs) has been extensively studied. However, the mechanism by which disease-related alterations in MSCs affect the immune response has not been reported.</p> Methods and results <p>MSCs were isolated from adipose tissue of healthy and EAE-induced mice and cultured with or without preconditioning usinglipopolysaccharide (LPS, 10 ng/mL). The immunomodulatory effects of MSC-conditioned media (CM) on the recall responses of splenocytes were evaluated. Cytokine levels, including IL-10, TGF-β, IL-17a, and nitric oxide (NO), were measured in the culture supernatants using enzyme-linked immunosorbent assay (ELISA) kits. The expression levels of transcription factors <i>Foxp3</i>, <i>Gata3</i>, <i>Rorc</i>, IFN-γ, and <i>Tbx21</i> in the splenocytes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). MSCs from healthy mice produced higher levels of IL-10 than those from EAE mice, with no significant difference in TGF-β production. Splenocytes treated with MSC-CM from healthy mice had higher <i>Foxp3</i> and <i>Gata3</i> expression, along with increased IL-10 and NO production, than those treated with EAE MSC-CM. In contrast, splenocytes treated with EAE MSC-CM showed higher Rorc expression and IL-17a levels. LPS preconditioning of EAE MSCs partially restored their immunomodulatory capacity, increased IL-10 levels, and decreased IL-17a levels. However, IFN-γ and <i>Tbx21</i> expression levels remained similar in splenocytes treated with MSC-CM from both healthy and EAE mice.</p> Conclusion <p>MSCs from EAE mice showed a reduced capacity to upregulate <i>Foxp3</i> mRNA and modulate immune responses in restimulated splenocytes compared to healthy MSCs. LPS preconditioning partially restored immunomodulatory properties.</p> Clinical trial number <p> Not applicable.</p>