Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia which are mechano- and osmo-sensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemo-sensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia, and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y₁₂ purinergic receptor, adenylyl cyclases (i.e., AC4, AC6 and AC8), protein kinase A (i.e., PKA RI- and PKA RII- regulatory subunits), exchange protein directly activated by cAMP isoform 2 (i.e., EPAC2), and A-kinase anchoring protein (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y₁₂ receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture, induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-S, a non-hydrolyzed analog of ATP, but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y₁₂ siRNAs suggesting that cilia and ciliary P2Y₁₂ are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemo-sensory organelles that detect biliary nucleotides through ciliary P2Y₁₂ receptors and transduce corresponding signals into a cAMP response.