Dysregulation of Hedgehog signaling can lead to several pathologies such as congenital defects and cancer. Here, we show that Hedgehog signaling is active in undifferentiated 3T3-L1 cells and decreases during adipocyte differentiation. Interestingly, this is paralleled by a decrease in Indian Hedgehog expression. We then tested if this down-regulation was sufficient to induce adipocyte differentiation. To this end, we demonstrate that the well-characterized Hedgehog inhibitor cyclopamine induced a decrease in Hedgehog signaling, similar to the one observed during adipocyte differentiation. However, cyclopamine did not induce nor potentiate adipocyte differentiation, as monitored by triglyceride staining and by the expression of several adipocyte markers: aP2, adipsin, C/EBPalpha, and Pref-1. Moreover, cyclopamine cannot substitute for other components of the differentiation medium: insulin, dexamethasone or IBMX. These results indicate that although Hedgehog signaling decreases during adipocyte differentiation, this down-regulation is not sufficient to trigger adipocyte differentiation. This suggests that Hedgehog signaling is an inadequate pharmacological target for patient suffering from syndromes associated with a decrease in fat mass, such as the ones observed in lipodystrophies.