Objective: Our aim in this study was to evaluate the levels of Activating Transcription Factor-6 (ATF-6), Inositol-Requiring Enzyme 1 (IRE-1), and C/EBP-Homologous Protein (CHOP), which are critical markers of endoplasmic reticulum (ER) stress, in patients with gestational diabetes mellitus (GDM), a condition increasingly recognized for its complex biochemical and molecular underpinnings. Materials and Methods: A total of 89 patients aged 18–45 years who applied to our hospital between January 2024 and December 2024 were included in our study. Of these, 45 patients were diagnosed with GDM during their routine pregnancy follow-up, while 44 were healthy pregnant women without any additional metabolic or obstetric complications. Serum samples were analyzed for ATF6, IRE-1, and CHOP levels, which are biochemical markers associated with ER stress. The data obtained were compared between the two groups. Results: The patients with GDM were older than those in the control group (31.98 ± 5.29 vs. 27.55 ± 5.92 years, P < 0.001). The mean number of gravidas (3.96 ± 2.01 vs. 2.48 ± 1.62, P < 0.001) and parity (2.18 ± 1.45 vs. 1.14 ± 1.07, P < 0.001) were significantly higher in the GDM group than in the control group. All GDM patients showed elevated levels of hemoglobin A1c (HbA1C) (6.69 ± 1.43% or 49.24 ± 15.25 mmol/mol, n = 45), indicating chronic hyperglycemia. GDM patients had elevated levels of red blood cells (RBC), hematocrit (HCT), mean platelet volume (MPV), platelet distribution width (PDW), red cell distribution width-coefficient of variation (RDW-CV) and potassium. They also had decreased glucose levels. Analysis revealed that there was no significant change in CHOP and IRE-1 levels in the GDM women compared with patients in the control group. However, the level of ATF-6 was significantly reduced in the GDM group compared with the control group (5.30, interquartile range [IQR] 4.59–6.28 vs. 5.92, IQR 5.17–8.43; P = 0.040). Despite no significant changes in CHOP and IRE-1 levels, the reduced ATF-6 levels in GDM patients highlight its potential as a specific biomarker for ER stress in the context of GDM. Conclusion: This study identifies ATF-6 as a potential biomarker for GDM, with reduced levels significantly associated with GDM and key clinical parameters. Our findings support the integration of ER stress and hematological markers into future research and clinical practice for GDM.