Adipose tissue-derived mesenchymal stem cells (AD-MSCs) have been shown to be capable of differentiating into multiple cell type and exert immunomodulatory effects. Since the selection of ideal stem cell is apparently crucial for the outcome of experimental stem cell therapies, therefore, in this study we compared AD-MSCs conditioned media (CM) from BALB/c, C57BL/6, and DBA mouse strains. No significant difference was found in the morphology, cell surface markers, in vitro differentiation and proliferation potentials of AD-MSCs isolated from C57BL/6, BALB/c, and DBA mice. The immunological assays showed some variation among the strains in the cytokines, nitric oxide (NO), and indoleamine 2,3-dioxygenase (IDO) production and immunomodulatory effects on splenocytes functions. Our results indicated a suppression of splenocytes proliferation in the presence of AD-MSC CM from the three inbred mouse strains. However, BALB/c CM exerted a higher suppression of splenocytes proliferation. AD-MSCs isolated from C57BL/6 and BALB/c mice produced higher levels of TGF-β than those from DBA mice. Furthermore, IL-17 and IDO production was higher in AD-MSCs isolated from BALB/c mice. Our results indicated an increased production of TGF-β, IL-4, IL-10, NO, and IDO by splenocytes in response to CM from BALB/c AD-MSCs. In conclusion, our results showed that the immunomodulatory properties of mouse AD-MSCs is strain-dependent and this variation should be considered during selection of appropriate stem cell source for in vivo experiments and stem cell therapy strategies. J. Cell. Biochem. 114: 955–965, 2013. © 2012 Wiley Periodicals, Inc.