Gemcitabine is the first-line treatment for pancreatic ductual adenocarcinoma (PDAC) as well as acts against a wide range of other solid tumors. Patients usually have a good initial response to gemcitabine-based chemotherapy but would eventually develop resistance. To improve survival and prognosis of cancer patients, better understanding of the mechanisms responsible for gemcitabine resistance and discovery of new therapeutic strategies are in great need. Amounting evidence indicate that the developmental pathways, such as Hedgehog (Hh), Wnt and Notch, become reactivated in gemcitabine-resistant cancer cells. Thus, the strategies for targeting these pathways may sensitize cancer cells to gemcitabine treatment. In this review, we will summarize recent development in this area of research and discuss strategies to overcome gemcitabine resistance. Given the cross-talk between these three developmental signaling pathways, designing clinical trials using a cocktail of inhibitory agents targeting all these pathways may be more effective. Ultimately, our hope is that targeting these developmental pathways may be an effective way to improve the gemcitabine treatment outcome in cancer patients.