Gemcitabine‑based chemotherapy is one of the most effective and commonly used chemotherapeutic regimens for biliary tract cancer (BTC). However, development of resistance to this drug limits its efficacy. The present study aimed to explore the effects of midkine (MDK) on the resistance of BTC cells to gemcitabine. Cell viability and proliferation were measured by a Cell Counting Kit‑8 assay and 5‑ethynyl‑2'‑deoxyuridine staining, respectively. Western blot analysis was used to detect the expression of E‑cadherin and vimentin. The results indicated that BTC cell lines were more resistant to gemcitabine plus MDK compared with gemcitabine alone. In terms of the underlying mechanism, MDK promoted the epithelial to mesenchymal transition (EMT) of BTC cells and the enhancing effect of MDK on gemcitabine resistance was abrogated when the EMT was blocked with small interfering (si)RNA targeting Twist. In addition, MDK promoted the expression of Notch‑1, while knockdown of Notch‑1 by siRNA blocked the EMT process in the BTC cell lines. Taken together, these results indicated that MDK promoted gemcitabine resistance of BTC through inducing EMT via upregulating Notch‑1. It was suggested that inhibition of the EMT is a promising strategy to overcome MDK‑induced drug resistance.