Sonic hedgehog (Shh) signaling critically regulates embryogenesis and tissue homeostasis. Here, we investigated the role of Shh signaling in mediating epithelial-mesenchymal transition (EMT) in human renal tubular epithelial cells HKC-8. Our RT-PCR assays demonstrated that TGF-β1 induced time-dependent changes in the mRNA transcript levels of Shh, with a steady rise from one hour post TGF-β1 treatment and a peak at four hours post TGF-β1 treatment. Furthermore, TGF-β1 induced a time-dependent increase in the mRNA transcript levels of Gli1. Pre-treatment with 2 or 5 µM cyclopamine significantly attenuated TGF-β1-induced rise in the mRNA transcript levels of Gli1, but failed to attenuate TGF-β1-induced rise in Shh mRNA transcript levels. Additionally, immunoblotting assays and immunofluorescence staining demonstrated that inhibition of Shh signaling by cyclopamine significantly attenuated TGF-β1-induced increase in the mRNA transcript levels of α-SMA, collagen I, and fibronectin. Gli1 overexpression induced Snail1 expression. Moreover, Gli(-/-) mice that had undergone unilateral ureteral obstruction for seven days showed significant reduction in the mRNA transcript levels of Snail1 compared to the wildtype controls. In conclusion, the current study provides novel insight into the regulation of EMT by the Shh/Gli1 signaling pathway, suggesting a critical role of Shh/Gli1 signaling in EMT of human renal tubular epithelial cells.