Purpose <p>Recurrent spontaneous abortion (RSA) involves complex pathophysiology, making it difficult to develop effective treatments and causing significant health and economic burdens. This study evaluated miR-363-5p as a potential biomarker for RSA and explored its role in disease progression to provide insights for clinical management.</p> Methods <p>A total of 68 serum samples from RSA patients and 97 from healthy pregnant controls were analyzed to determine miR-363-5p expression and its clinical significance. The EVT cell line HTR-8/SVneo was used for in vitro experiments. Cell proliferation was assessed using CCK-8 assays, while Transwell assays evaluated migration and invasion. qRT-PCR detected miR-363-5p and S100A1 expression levels. Dual-luciferase reporter assays confirmed the interaction between miR-363-5p and S100A1.</p> Results <p>miR-363-5p expression was significantly lower in RSA patients than in healthy pregnant controls. ROC analysis indicated its high diagnostic potential for RSA. In vitro experiments showed that miR-363-5p promoted HTR-8/SVneo cell proliferation, migration, and invasion while inhibiting apoptosis. S100A1 was identified as a direct target of miR-363-5p in RSA. miR-363-5p regulates EVT cell functions by suppressing S100A1 expression.</p> Conclusions <p>Serum miR-363-5p downregulation may serve as a diagnostic biomarker for RSA. miR-363-5p likely affects pregnancy outcomes in RSA by targeting S100A1 to regulate EVT cell functions. These findings suggest that miR-363-5p has potential for both diagnosing and treating RSA.</p>