Gallbladder cancer (GBC) is a particularly deadly type of cancer with a 5-year survival rate of only 10%. New effective therapeutic strategies are greatly needed. Recently, we have shown that Hedgehog (Hh) signaling is reactivated in various types of cancer and is a potential therapeutic target. However, little is known about the biological significance of Hh signaling in human GBC. In this study, we determined whether Hh signaling could be a therapeutic target in GBC. The Hh transcription factor Gli1 was detected in the nucleus of GBC cells but not in the nucleus of normal gallbladder cells. The expression levels of Sonic Hh (Shh) and Smoothened (Smo) in human GBC specimens (n = 37) were higher than those in normal gallbladder tissue. The addition of exogenous Shh ligand augmented the anchor-dependent and anchor-independent proliferation and invasiveness of GBC cells in vitro. In contrast, inhibiting the effector Smo decreased the anchor-dependent and anchor-independent proliferation. Furthermore, the suppression of Smo decreased GBC cell invasiveness through the inhibition of MMP-2 and MMP-9 expression and inhibited the epithelial–mesenchymal transition. In a xenograft model, tumor volume in Smo siRNA-transfected GBC cells was significantly lower than in control tumors. These results suggest that Hh signaling is elevated in GBC and may be involved in the acquisition of malignant phenotypes, and that Hh signaling may be a potential therapeutic target for GBC.