Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling, the dysregulation of which causes several pathologies, such as congenital defects and cancer, is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells, such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis, but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells, the maturation process, but not the early steps of human mesenchymal stem cell differentiation, is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein alpha and peroxisome proliferator-activated receptor (PPAR) gamma2 expression, whereas PPARgamma1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes, adipogenesis appears dramatically impaired, with reduced lipid accumulation, a decrease in adipocyte-specific markers, and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells.