[Objective] To investigate the ability of enterovirus A71 (EV-A71) to traverse the blood-brain barrier (BBB) and infect human cerebral organoids, as well as the impact of EV-A71 on BBB integrity following retrograde invasion into the central nervous system via neural routes, and to characterize the infection profiles of EV-A71 in the BBB and human cerebral organoids and the associated inflammatory responses. [Methods] An invitro BBB model was established by seeding human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes into a Transwell system. Human cerebral organoids were generated from human induced pluripotent stem cells (hiPSCs), and a BBB-human cerebral organoid co-culture model was subsequently established. EV-A71 at three titers (10², 10⁴, and 10⁶ TCID₅₀) was used to infect each cell type of the BBB to evaluate viral replication kinetics and cytopathic effects (CPE) across different cell types. Subsequently, the BBB-human cerebral organoid co-culture model was infected with 10⁴ TCID₅₀ EV-A71 using two distinct routes: (1) an apical inoculation strategy, in which EV-A71 was directly applied to the human brain microvascular endothelial cell layer; and (2) a retrograde infection strategy, in which human cerebral organoids were first infected and subsequently co-cultured with the BBB model. Viral RNA copy numbers in culture supernatants and cells were quantified by RT-qPCR. BBB integrity was assessed by measuring transendothelial electrical resistance, and inflammatory responses were evaluated by determining the expression levels of inflammatory cytokines in human cerebral organoids using RT-qPCR. [Results] EV-A71 efficiently replicated in human brain microvascular endothelial cells, brain vascular pericytes, and astrocytes, inducing pronounced CPE. Among these cell types, astrocytes were the most susceptible to EV-A71 infection, showing the most rapid progression of cytopathic changes, followed by brain vascular pericytes. Human brain microvascular endothelial cells were relatively less susceptible, requiring higher viral titers to establish effective infection. Under apical inoculation conditions, EV-A71 replicated within the BBB, compromised BBB integrity, traversed the barrier to infect human cerebral organoids, and significantly activated inflammatory responses. In the retrograde infection model, EV-A71 replicated within human cerebral organoids, preferentially targeted neurons and astrocytes, triggered cellular inflammatory responses, and subsequently invaded the BBB in a retrograde manner, resulting in compromised structural integrity of the BBB. [Conclusion] EV-A71 can traverse the BBB to infect human cerebral organoids and activate inflammatory responses, and can also infect human cerebral organoids first and subsequently disrupt BBB integrity in a retrograde manner. This study systematically characterizes the infection dynamics of EV-A71 in an invitro BBB-human cerebral organoid model, provides a robust experimental model for in-depth analysis of the mechanisms underlying EV-A71 invasion of the central nervous system, and lays a research foundation for future investigations into viral pathogenesis and antiviral therapeutic development.