TGF-β is a cytokine implicated in multiple cellular responses, including cell cycle regulation, fibrogenesis, angiogenesis and immune modulation. In response to pro-inflammatory and chemotactic cytokines and growth factors, cholangiocytes prime biliary damage, characteristic of cholangiopathies and pathologies that affect biliary tree. The effects and signaling related to TGF-β in cholangiocyte remains poorly investigated. In this study, the cellular response of human cholangiocytes to TGF-β was examined. Wound-healing assay, proliferation assay and cell cycle analyses were used to monitor the changes in cholangiocyte behavior following 24 and 48 h of TGF-β stimulation. Moreover, proteomic approach was used to identify proteins modulated by TGF-β treatment. Our study highlighted a reduction in cholangiocyte proliferation and a cell cycle arrest in G0/G1 phase following TGF-β treatment. Moreover, proteomic analysis allowed the identification of four downregulated proteins (CaM kinase II subunit delta, caveolin-1, NipSnap1 and calumin) involved in Ca2+ homeostasis. Accordingly, Gene Ontology analysis highlighted that the plasma membrane and endoplasmic reticulum are the cellular compartments most affected by TGF-β. These results suggested that the effects of TGF-β in human cholangiocytes could be related to an imbalance of intracellular calcium homeostasis. In addition, for the first time, we correlated calumin and NipSnap1 to TGF-β signaling.