Background: Gemcitabine is among the most commonly utilized chemotherapeutic agents for treating pancreatic cancer (PC), yet patients ultimately develop chemoresistance and thus exhibit a poor prognosis. Long noncoding RNAs (lncRNAs) can function as key regulators of PC progression and may serve as prognostic biomarkers in individuals with gemcitabine-resistant PC. This study sought to explore the role of the lncRNA DBH-AS1 in this oncogenic setting. Methods: Based on public databases and qRT-PCR analyses the expression of lncRNA DBH-AS1 in PC tissues and cell lines. The effects of lncRNA DBH-AS1 on proliferation and gemcitabine resistance were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA DMH-AS1, miR-3163 and USP44. Results: We found that PC tissues exhibited DBH-AS1 downregulation that was particularly pronounced in gemcitabine-resistant PC tissues and cells. This DBH-AS1 downregulation was negatively correlated with the malignancy of PC tumors and with patient survival outcomes. Additionally, decreased DBH-AS1 expression in PC was found to be linked to the METTL3-dependent m(6)A methylation of the lncRNA, with functional analyses revealing that DBH-AS 1 was able to suppress the growth of PC cells. Mechanistically, DBH-AS1 was able to increase PC cell sensitivity to gemcitabine by sequestering miR-3163 and thus upregulating USP44 in these tumor cells. Clinically, patient-derived PC tumor xenografts exhibiting high levels of DBH-AS I expression were found to be responsive to gemcitabine treatment. Conclusions: Overall, these data underscore a key role for DBH-AS1 as a regulator of PC tumor growth and a promising therapeutic target capable of predicting PC patient responsiveness to gemcitabine treatment.