The properly programmed “stemness” of tissue specific stem cells, including adipose tissue stem cells (ASCs), is the core feature for their function of tissue regeneration. Although ASCs harbor great potential for clinical application, it is poorly understood how ASCs respond to obesity, an affliction now affect over one third of the national population and health risks of cardiovascular diseases. We recently identified a set of cell surface markers to enrich ASCs from murine visceral adipose tissue. We observed that adipose tissue stromal cells bearing CD45-CD31-Sca-1+CD49f-CD34+ were highly enriched for cells with sphere-initiation and multilineage differentiation capacity. Interestingly, the frequency of ASCs was markedly reduced in obese visceral stroma and their capability to derive multilineage progeny were severely impaired, compared to those in lean mice. Given the heterogeneity of isolated ASCs, we performed single cell sequencing analysis to understand mechanisms underlying the impaired “stemness” of obese ASCs. Freshly isolated ASCs from obese or age matched lean mice were subjected to single cell library generation (10X Genomics), followed by high-throughput sequencing. An average of 5500 UMI (unique molecular index) were detected in each cell and a total of 18,835 genes were detected in each sample (mapped to mm10). Interestingly, ASCs isolated from lean or obese adipose tissue displayed distinct pattern after K-mean clustering. Further, functional analysis revealed that critical networks in ASCs are significantly altered under obese conditions, including suppressed adipogenic program initiation and impaired impaired antioxidative defense. Further, multiple signaling networks triggered by both innate and adaptive immune responses displayed high activation Z-score in obese ASCs than that in lean ASCs. Overall, our results suggested that obesity severely impaired ASCs “stemness”, which may be attributed to multiple factors in obese adipose tissue, including the proinflammatory microenvironment. Further analyses of these profiles will provide critical information on how obesity alters the “stemness” program in ASCs and how to “revive” obese ASCs tissue regenerative ability.