Hedgehog (Hh) signaling is a critical pathway essential for embryonic development and maintaining adult tissue homeostasis. Dysregulation of this pathway is linked to numerous developmental disorders and various cancers. In vertebrates, Hh signal transduction is tightly dependent on the primary cilium, a small antenna-like organelle on the cell surface. Upon activation of the pathway, Smoothened (Smo), an atypical G protein-coupled receptor (GPCR), is released from the inhibitory control of Patched (Ptch) and translocates into the primary cilium, a pivotal step for Smo activation of downstream signaling, as discussed Chapter 1. Despite its importance, the molecular mechanisms governing Smo translocation and regulation remain inadequately understood.To address this, I leveraged the TurboID proximity labeling tool a method not previously used to study Smo signaling in the context of Hh pathway regulation. Chapter 2 details the development of Smo-TurboID system designed to label proteins that are proximal to Smo during its translocation into the primary cilium over the course of Hh signaling activation. Using Tandem Mass Tag (TMT)-mediated quantitative mass spectrometry, my work shows that Smo-TurboID system not only identified several well-established Hh signaling proteins, but also uncovered novel transient changes in the local micro-environment near Smo. In Chapter 3, my work focused on the newly identified Smo-interacting proteins, including the Catalytic subunit of Protein Kinase A (PKA-C) , which associates with Smo following Hh signaling activation, and G protein-coupled receptor kinase 2 (Grk2), a putative transient interactor. Notably, inhibition of Smo or Grk2 in Hh pathway diminishes the interaction between Smo and PKA-C, confirming the necessity of Smo-Grk2-PKA axis in Hh signaling. Chapter 4 extends this work by identifying GRK-interacting protein 1(Git1) as a positive regulator in Hh pathway. Using the Smo-TurboID system, Git1 emerged as a high-priority candidate, localizing at the base of primary cilium. CRIPSR/Cas9 knockout of Git1 in fibroblast markedly impaired Hh signaling and a reduction of phosphorylated Smo in the primary cilium. Importantly, I identified a critical link between Git1 and Grk2, a well-established activator that phosphorylates Smo within the cilia, offering fresh insight into the fine-tuning of Hh pathway regulation. Collectively, these findings provide a broader view of molecular regulation of Smo in Hh pathway at the primary cilium. By utilizing Smo-TurboID system, my work identified both known and novel Smo-associated proteins, revealing dynamic changes near Smo during it signaling transduction. Additionally, my work establishes the connection between Smo, Git1 and Grk2, offering valuable insights into potential therapeutic strategies targeting Smo interactors for Hh signaling-related cancers.