Objective <p>Recurrent spontaneous abortion (RSA) is a major pregnancy complication with unclear pathogenesis. This study aims to investigate the role of miR-20b-5p and its downstream target HIF-1α in trophoblast function and RSA pathogenesis.</p> Methods <p>Placental villous tissues from uRSA patients and healthy pregnant women were collected to assess trophoblast migration, invasion, apoptosis, and placental angiogenesis. The expression levels of miR-20b-5p and HIF-1α were analyzed using qPCR, Western blot, and immunohistochemistry. HTR-8/SVneo cells were transfected with miR-20b-5p mimics, si-miR-20b-5p, and si-HIF-1α to evaluate their effects on trophoblast function. Luciferase reporter assays were performed to confirm the regulatory relationship between miR-20b-5p and HIF-1α.</p> Results <p>Trophoblast cells isolated from uRSA patients exhibited impaired trophoblast invasion and increased trophoblast apoptosis, accompanied by increased trophoblast apoptosis. miR-20b-5p was significantly upregulated, whereas HIF-1α expression was downregulated in uRSA placental tissues. Overexpression of miR-20b-5p inhibited trophoblast migration, invasion, and endothelial-like differentiation, while its knockdown enhanced these functions. HIF-1α was identified as a direct target of miR-20b-5p, and its knockdown partially reversed the effects of miR-20b-5p inhibition.</p> Conclusion <p>miR-20b-5p negatively regulates trophoblast function by targeting HIF-1α, contributing to trophoblast dysfunction and RSA pathogenesis. The miR-20b-5p/HIF-1α axis may serve as a potential therapeutic target for RSA.</p>