Background <p>Pancreatic cancer is a malignant disease with a poor prognosis. Gemcitabine (GEM), the first-line treatment drug, shows limited efficacy because of the notorious drug resistance of pancreatic cancer. Therefore, the development of sensitive drugs for pancreatic cancer is essential. AZD5153 is a novel bivalent BET bromodomain inhibitor with multiple anti-tumor effects on malignancy. Here, we aimed to investigate the effect of AZD5153 on the GEM sensitivity in human pancreatic cancer cells.</p> Methods <p>Sulforhodamine B (SRB), clone formation assays were designed to characterize the cell viability and clone formation after treatment with AZD5153 and/or GEM. DAPI staining, flow cytometry and western blotting were used to identify the cell apoptosis. RNA-seq analysis, western blotting and qPCR were also conducted to confirm the signaling pathway involved in it. Nude mice bearing PANC-1 pancreatic cancer xenograft model was conducted to confirm the combination effect of GEM and AZD5153 in vivo.</p> Results <p>As a result, AZD5153 presented a strong anti-proliferation activity and exerted synergistic effects when combined with GEM in BXPC3 and PANC-1 cell lines.Meanwhile, the combination treatment also inhibited colony formation in these two cell lines. Additionally, AZD5153 combined with GEM induced cell apoptosis. Further investigations revealed that the combination of AZD5153 and GEM decreased the phosphorylation of ERK/mTOR signaling proteins, the specific chemical activators PDBu (activator of ERK) reversed the expression of c-PARP. Besides, the expression of MUC2 was remarkable decreased after combination treatment.</p> Conclusion <p>In conclusion, these results suggested that AZD5153 might be an excellent GEM sensitizer in pancreatic cancer.</p>