Background <p>Inflammatory cytokines are crucial for various cellular mechanisms, including differentiation, fibrosis, and proliferation. Cytokine dysregulation provokes pathophysiologic conditions in cholangiocytes, even cholangiocarcinogenesis. Specifically, transforming growth factor-beta (TGF-β) and interleukin-6 (IL-6) are known to be related to cholangiocarcinoma progression. We investigated the pathophysiological signaling in normal cholangiocyte (H69) spheroids mediated by TGF-β1 or/and IL-6.</p> Methods and results <p>The spheroids were treated with TGF-β1 or/and IL-6 for 1 and 3 days, and then the secretion levels of TGF-β1 and IL-6, as well as their mediated protein expression levels, were measured. TGF-β1-mediated IL-6 signaling was assessed using TGF-β1- and IL-6-specific small interfering RNA (siRNA). The secretion of IL-6 and the expression of its receptor were significantly increased in all treated spheroids at 3 days. Treatment of spheroids with TGF-β1 or TGF-β1 plus IL-6 increased the expression of epithelial–mesenchymal transition (EMT)- and fibrosis-related proteins, but IL-6 treatment alone did not. Moreover, all treated spheroids showed a marked increase in size and the expression levels of proliferation-related proteins. Furthermore, the levels of IL-6 secretion and proliferation-related protein expression remained unchanged in TGF-β1 or/and IL-6 siRNA transfectants compared with those of the scrambled siRNA transfectant.</p> Conclusions <p>Our findings suggest that long-term exposure to TGF-β1 triggers the malignant transformation of cholangiocytes by activating EMT and fibrosis, as well as IL-6/STAT3-mediated proliferation signaling.</p>