Objective To investigate the potential effects of glycemic fluctuation on copper homeostasis and blood–brain barrier （BBB） function in hCMEC/D3 human brain microvascular endothelial cells. Methods hCMEC/D3 cells were conventionally cultured and divided into a control group （glucose 25 mmol/L for 4 days） and a fluctuating glucose group （FG group： glucose 25 mmol/L for 8 h followed by glucose 55 mmol/L for 8 h， repeatedly for 4 days）. Cell viability was assessed using the CCK-8 assay， and cell morphology was observed under an light microscope. A Transwell system was used to construct a monolayer cell model， and transendothelial electrical resistance （TEER） was measured daily. Monolayer permeability was evaluated using the phenol red assay. Intracellular copper levels and reactive oxygen species （ROS） levels were detected. The mRNA expression levels of copper transporter 1 （SLC31A1）， copper-transporting ATPase β chain （ATP7B）， cytochrome c oxidase copper chaperone 17 （COX17）， and antioxidant 1 copper chaperone （ATOX1） were determined by qPCR. The protein expression levels of SLC31A1， ATP7B， COX17， ATOX1， zonula occludens-1 （ZO-1）， occludin， and Claudin-1 were analyzed by Western blot. Results Cell viability in the FG group was significantly lower than that in the control group （P < 0.05）. Cells in the control group exhibited robust growth and formed a dense network with extensive intercellular connections， whereas cell growth in the FG group was inhibited， with markedly reduced cell numbers and diminished intercellular contacts. The TEER value in the FG group was significantly lower than that in the control group （P < 0.05）， and the phenol red permeability assay revealed increased permeability and disrupted monolayer integrity in the FG group （P < 0.001）. Both the copper ion detection kit and copper-specific fluorescent probe showed a significant reduction in intracellular copper levels in the FG group. Intracellular ROS levels were markedly elevated on day 4 in the FG group （P < 0.05）. qPCR results demonstrated that， compared with the control group， the mRNA expression of ATOX1， COX17， and SLC31A1 was significantly decreased， whereas ATP7B expression was significantly increased （P < 0.05）. Western blot analysis showed that the protein expression of ZO-1， occludin， and Claudin-1 was significantly reduced in the FG group （P < 0.05）. The protein expression of SLC31A1 （P < 0.05）， ATOX1， and COX17 was decreased， while ATP7B expression was increased （P < 0.05）. Immunofluorescence analysis further confirmed that， compared with the control group， SLC31A1 expression was reduced and ATP7B expression was elevated in the FG group（P < 0.05）. Conclusion Glycemic fluctuation may impair the structure and function of the BBB in hCMEC/D3 cells by inducing damage to barrier-related structures， increasing cellular permeability， and promoting ROS accumulation. This pathological process may be closely associated with intracellular copper homeostasis imbalance， providing new insights into the mechanisms underlying BBB dysfunction in conditions characterized by glycemic instability.