Background <p>Hand, foot, and mouth disease (HFMD) is a widespread infectious disease primarily affecting infants and young children. Enterovirus A71 (EV-A71) comprises eight genogroups, among which subgroup C4 is the dominant viral agent in China and is frequently associated with HFMD and central nervous system infections. The genetic characteristics of an EV-A71 subgroup C4 strain obtained in this study were analyzed using whole-genome sequencing. Its biological characteristics, including infectivity, replication, and cytotoxicity, were investigated in human rhabdomyosarcoma (RD) and African green monkey kidney (Vero) cells.</p> Methods <p>A clinical EV-A71 C4 subgourp GD10 strain isolated in China was examined to evaluate its genetic and biological features. Its relationships with strains listed in GenBank were evaluated using phylogenetic analysis. Viral infectivity and replication were assessed in RD and Vero cells. Cytotoxicity was evaluated by measuring cell viability, lactate dehydrogenase (LDH) release, and ATP levels. Effects on blood–brain barrier (BBB) integrity were investigated in vitro by assessing transendothelial electrical resistance and viral load across the barrier.</p> Results <p>Sequence analysis confirmed that GD10 belonged to subgroup C4 and closely resembled strains from China. GD10 infection induced a pronounced cytopathic effect and elevated viral RNA levels in RD cells but not in Vero cells. The infection time-dependently increased LDH release and reduced ATP levels. GD10 compromised BBB integrity and crossed the cellular barrier in vitro.</p> Conclusion <p>The GD10 strain demonstrated strong adaptability to RD cells and impaired BBB function. Our results improve the understanding of virus–host interactions and may support efforts towards EV-A71 vaccine development.</p> Clinical Trial <p>Not applicable</p>