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Background: Gestational diabetes mellitus (GDM) is characterized by pregnancy-induced insulin resistance and β-cell dysfunction and is increasingly recognized as a state of cardiometabolic and endothelial dysregulation. Salusin-β, a bioactive peptide implicated in vascular inflammation and metabolic disorders, may play a role in GDM pathophysiology. However, data regarding its clinical relevance in GDM remain limited. Objective: This study aimed to compare serum salusin-β levels between women with GDM and healthy pregnant controls and to evaluate its diagnostic performance. Methods: This prospective study included 144 pregnant women between 24 and 28 weeks of gestation (70 with GDM and 74 healthy controls). GDM was diagnosed using a 75-g oral glucose tolerance test according to American Diabetes Association criteria. Serum salusin-β levels were measured using ELISA. Between-group comparisons were performed using appropriate parametric or non-parametric tests. Multivariable logistic regression analysis was performed to evaluate the independent association between salusin-β and GDM. Sensitivity analyses using an expanded adjustment model were also conducted. Diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis. Results: Salusin-β levels were significantly lower in women with GDM compared to controls (68.53 [53.05-84.27] vs. 128.03 [76.74-261.35] pg/mL; p < 0.001). In the expanded multivariable logistic regression analysis, lower serum salusin-β levels remained independently associated with GDM (OR = 0.990, 95% CI: 0.985-0.996, p = 0.001). ROC analysis demonstrated acceptable discriminatory performance (AUC = 0.754, 95% CI: 0.672-0.833). The optimal cut-off value of 87.1 pg/mL yielded 80.0% sensitivity and 66.2% specificity. Conclusions: Serum salusin-β levels are significantly reduced in women with GDM and independently associated with disease presence. Although not sufficient as a standalone diagnostic marker, salusin-β demonstrates moderate discriminatory ability and may serve as a complementary biomarker reflecting vascular and metabolic dysregulation in GDM.