Background: Gestational diabetes mellitus (GDM) is characterized by elevated blood glucose during pregnancy, which may affect both the fetus and the pregnant woman. This study introduced the expression and regulatory ability of long non-coding RNA (lncRNA) DLX6 Antisense RNA 1 (DLX6-AS1) in patients with GDM, aiming to reveal the action potential and diagnostic value of DLX6-AS1. Methods: This study included 70 pregnant patients with GDM and 50 healthy pregnant women. DLX6-AS1 levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR), and the diagnostic value of DLX6-AS1 was evaluated by receiver operating characteristic (ROC) curve. The GDM cell model was constructed using human chorionic trophoblast cells, and the cell proliferation capacity was assessed using cell counting kit-8 (CCK-8) method. Cell apoptosis was analyzed by flow cytometry. Moreover, luciferase assay was performed to evaluate the relationship between DLX6-AS1 and miR-497-5p. Results: DLX6-AS1 and blood glucose levels were markedly increased in GDM patients, and a positive correlation was observed between both levels (r = 0.7072, p 0.0001). GDM affected the cell activity, while DLX6-AS1 silencing enhanced the proliferation activity, and suppressed cell apoptosis in GDM cell model via directly targeting miR-497-5p. miR-497-5p expression was low in GDM, and its content was affected by DLX6-AS1 silencing (p 0.001). Furthermore, DLX6-AS1 exhibited a promising diagnostic function in GDM (area under the curve (AUC) = 0.937, sensitivity = 92.9%, specificity = 86.0%). Conclusions: DLX6-AS1 was positively expressed and mediated GDM through sponge miR-497-5p, suggesting it may be used as a diagnostic factor to predict the occurrence of GDM.