Cancer-associated fibroblasts (CAFs) are the dominant component of the tumor microenvironment (TME) that can be beneficial to the generation and progression of cancer cells leading to chemotherapeutic failure several mechanisms. Nevertheless, the roles of CAFs on anti-cancer drug response need more empirical evidence in cholangiocarcinoma (CCA). Herein, we examined the oncogenic roles of CAFs on gemcitabine resistance in CCA cells mediated IL-6/STAT3 activation. Our findings showed that CCA-derived CAFs promote cell viability and enhance gemcitabine resistance in CCA cells through the activation of IL-6/STAT3 signaling. High expression of IL-6R was correlated with a poor overall survival rate and gemcitabine resistance in CCA, indicating that IL-6R can be a prognostic or predictive biomarker for the chemotherapeutic response of CCA patients. Blockade of IL-6R on CCA cells by tocilizumab, an IL-6R humanized antihuman monoclonal antibody, contributed to inhibition of the CAF-CCA interaction leading to enhancement of gemcitabine sensitivity in CCA cells. The results of this study should be helpful for modifying therapeutic regimens aimed at targeting CAF interacting with cancer cells resulting in the suppression of the tumor progression but enhancement of drug sensitivity.