Adipose tissue contains abundant multipotent mesenchymal stem cells with strong proliferative and differentiating potential into adipocytes, osteocytes, and chondrocytes. However, adipose-derived mesenchymal stem cells (ASCs) showed variable characteristics based on the tissue-harvesting site. This study aimed at comparing human adipose-derived mesenchymal stem cell from the orbit (Orbital ASCs) and abdomen (Abdominal ASCs). Orbital and abdominal ASCs were isolated during an upper or lower blepharoplasty operation and liposuction, respectively. Flow cytometric analysis was done to analyze the surface antigens of ASCs, and cytokine profiles were measured using Luminex assay kit. The multilineage potential of both ASCs was investigated using Oil Red O, alizarin red, and alcian staining. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to measure mRNA levels of genes involved in these trilineage differentiations. Our results showed that both types of ASCs expressed the cell surface markers which are commonly expressed stem cells; however, orbital-ASCs showed higher expressions of CD73, CD90, CD105, and CD146 than abdominal ASCs. Unlikely, orbital-ASC expressed CD31, CD45 and HLA-DR lesser than abdominal-ASCs. Orbital ASCs secreted higher concentrations of eotaxin, fractalkine, IP-10, GRO, MCP-1, IL-6, IL-8, and RANTES but lower MIP-1, FGF-2, and VEGF concentrations than abdominal-ASCs. Our result showed that orbital ASCs have higher potential towards adipogenic and osteogenic differentiation but lower tendency to chondrogenesis when compared with abdominal ASCs. In conclusion, tissue-harvesting site is a strong determinant for characterization of adipose-derived mesenchymal stem cells. Understanding defining phenotypes of such cells is useful for making suitable choices in different regenerative clinical indications.