Objective: Pancreatic stone protein (PSP) is a secretory protein associated with systemic inflammation and β-cell stress. Given its reported association with type 2 diabetes mellitus, this study aimed to evaluate the relationship between serum PSP levels and gestational diabetes mellitus (GDM) and to assess its diagnostic performance. Methods: This single-center, prospective cohort study was conducted between June 2024 and May 2025. Eighty-four pregnant women at 24–28 weeks’ gestation were enrolled: 42 diagnosed with GDM according to Carpenter–Coustan criteria and 42 healthy controls. Serum PSP levels were measured using ELISA at the time of GDM diagnosis, and prior to treatment initiation. Group comparisons were performed using appropriate parametric and non-parametric tests. Diagnostic performance was evaluated by receiver operating characteristic (ROC) curve analysis. Results: Maternal weight was significantly higher in the GDM group (78.3 ± 10.0 kg vs. 68.4 ± 7.6 kg, p < 0.001). Serum PSP levels were significantly elevated in women with GDM compared to controls (8.89 ± 0.81 ng/mL vs. 7.72 ± 0.64 ng/mL, p < 0.001). Neonatal birth weight was also higher in the GDM group (3606 ± 507 g vs. 3355 ± 308 g, p = 0.008), while Apgar scores did not differ significantly. ROC analysis demonstrated an AUC of 0.883 (95% CI: 0.805–0.946, p < 0.001). At a cut-off value of 8.38 ng/mL, sensitivity was 76.2% and specificity was 85.7%. Conclusions: Serum PSP levels are significantly elevated in GDM and demonstrate good diagnostic performance. PSP may serve as a supportive biomarker reflecting β-cell stress in GDM, warranting further multicenter validation studies.