Obesity is a health problem characterized by large expansion of adipose tissue. During this expansion, genotoxic stressors can be accumulated and negatively affect the Mesenchymal Stem Cells (MSCs) of adipose tissue. Due to the oxidative stress generated by these genotoxic stressors, senescence phenotype might be observed in adipose tissue MSCs. Senescent MSCs lose their proliferations and differentiation properties and secrete senescence-associated molecules to their niche thus triggering senescence for the rest of the tissue. Accumulation of senescent cells in adipose tissue results in decreased tissue regeneration and functional impairment not only in the close vicinity but also in the other tissues. Here we hypothesized that declined tissue regeneration might be associated with loss of stemness in MSCs population. We analyzed the expression of several stemness genes in adipose tissue MSCs of high-fat diet and normal diet mice models. Since the MSCs population covers a small percentage of the pluripotent stem cells, a role in proliferation and tissue regeneration, we measured the percentage of these cells via TRA-1-60 surface antigen. Additionally, by conducting a shotgun proteomic approach using LC-MS/MS, whole cell proteome of the adipose tissue MSCs of high-fat diet and normal diet mice were analyzed and identified proteins were evaluated via Gene Ontology and PPI network analysis. MSCs of obese mice showed senescent phenotype and altered cell cycle distribution due to a hostile environment with oxidative stress in adipose tissue where they reside. Additionally, the number of pluripotent markers expressing cells declined in the MSC population of the high-fat diet mice. Gene expression analysis evidenced the loss of stemness with a decrease in the expression of stemness-associated genes. Of the proteomic comparison of the normal and the high-fat diet group, MSCs revealed that stemness-associated molecules were decreased while inflammation and senescence-associated phenotypes emerged in obese mice MSCs. Our results showed us that the MSCs of adipose tissue may lose their stemness properties due to obesity-associated stress conditions.