The membrane-bound protein Lymphocyte Antigen 6 Family Member D (LY6D) has been implicated in stemness and is associated with various tumors. LY6D is specifically expressed in cancer cells, with little expression in normal cells, but its physiological function in cancer progression remains poorly understood. Here, we investigated how LY6D was associated with pancreatic ductal adenocarcinoma (PDAC), and involved in stemness. We conducted functional analysis of LY6D to evaluate its contribution to impacting PDAC cells in vitro. Using our in-house developed stem cell separation technique, which can isolate cells with low proteasome activity and high stem cell properties, we successfully enriched cells with extremely high tumorigenic ability. We then used these cells, to perform in vitro functional assays by manipulating the LY6D expression. Immunohistopathological analysis was also conducted to reveal the clinical significance of LY6D in PDAC. In vitro functional assays demonstrated that LY6D was crucially involved in the cancer malignant phenotype including invasive ability, drug resistance, migration abilities, and cancer stemness. Immunohistopathological analysis revealed that high LY6D expression levels were associated with high recurrence rate and poorer prognosis in PDAC. Our present findings demonstrated that LY6D plays a key role in regulation of cancer stem cells. LY6D is not only a prognostic indicator but also could be a promising therapeutic target in PDAC.