Objective <p>We aimed to probe the effect of microRNA (miR)-455-3p derived from human umbilical cord mesenchymal stem cell exosomes (hucMSCs-Exos) in targeting calcium/calmodulin-dependent protein kinase 2 inhibitor 1 (CAMK2N1) to mitigate trophoblast cell injury in gestational diabetes mellitus (GDM).</p> Methods <p>hucMSCs were cultured, and Exos were isolated and characterized using transmission electron microscopy and Western blot (WB) for exosomal surface markers CD63, TSG101, and Calnexin. An in vitro GDM model was established by exposing human trophoblast cells (HRT-8/SVneo) to high glucose (HG), followed by intervention with Exos or overexpression of CAMK2N1. RT-qPCR or WB was applied to test miR-455-3p and CAMK2N1 expression levels. CCK-8 assay was adopted to assess cell proliferation, Transwell assay was applied to test cell migration, and flow cytometry was implemented to assess cell apoptosis. Bioinformatics websites and a dual-luciferase reporter gene assay were conducted to verify the targeting relationship between miR-455-3p and CAMK2N1.</p> Results <p>The successfully isolated Exos expressed the exosomal markers CD63 and TSG101. Treatment with hucMSCs-Exos and hucMSCs-Exos carrying miR-455-3p mimic enhanced the migration and proliferation of HG-induced HRT-8/SVneo cells while reducing cell apoptosis. In contrast, the miR-455-3p inhibitor and overexpression of CAMK2N1 reversed these protective effects, leading to decreased cell migration and proliferation and increased apoptosis. Furthermore, bioinformatics analysis and experimental validation confirmed that miR-455-3p directly targeted and negatively regulated CAMK2N1.</p> Conclusion <p>miR-455-3p derived from hucMSCs-Exos exerts a protective effect against trophoblast cell injury in GDM by targeting and downregulating CAMK2N1.</p>