Hedgehog (Hh) signaling plays key roles in animal development and tissue homeostasis. Binding of the secreted ligand to its Ptch1 receptor triggers Hh signaling through distinct canonical or noncanonical signaling pathways. Canonical Hh signaling leads to the activation of Gli transcription factors to induce Hh target-gene expression. In contrast, noncanonical Hh signaling regulates cytoskeleton rearrangement and apoptosis. Recently, it has been shown that primary cilia are important for canonical Hh signaling, but the ciliary role for signaling through the noncanonical pathway remains unresolved. Here, we examine the role of primary cilia in noncanonical Hh signaling in cultured mammalian cells. We found that Hh pathway activation in mouse embryonic fibroblast cells (MEFs) increases microtubule acetylation via smoothened (Smo), and suppression of Hh signaling by a Smo antagonist abrogates the microtubule acetylation. Using genetically engineered MEFs, we revealed that the increase in microtubule acetylation by Hh is dependent on Smo, but not on Sufu or Gli. In Kif3a(-/-) MEFs, which cannot form primary cilia, we observed that primary cilia were required for transducing noncanonical Hh signaling. Furthermore, we revealed that an increase in intracellular calcium is important for Hh-dependent tubulin acetylation at the downstream of Smo. Collectively, these findings suggest that Smo and primary cilia-dependent noncanonical Hh signaling leads to post-translational regulation of microtubules and may be important for modulating cell behaviors. (C) 2016 Elsevier Inc. All rights reserved.