Objective: This study aimed to investigate the relationship between sleep disorders and cardiac microvascular injury in myocardial ischemia-reperfusion injury (MI/RI) mice. Design: Mice were placed in a sleep deprivation chamber. The sweep bar moved along the bottom of the cage every 2 min during the light cycle (ZT0-12) and was stationary during the dark cycle (ZT12-24) for 16 weeks. Then, a MI/RI model was prepared to evaluate cardiac microvascular injury and investigate its specific mechanism via proteomics analysis. Results: Here, we showed that 16 weeks of sleep fragmentation (SF) exacerbated cardiac microvascular injury in MI/RI mice. Mechanistically, we found that SF promotes sympathetic overactivity, increases the level of norepinephrine (EPI) in plasma, thereby promotes the chemotaxis of neutrophils (NEs) and formation of Neutrophil extracellular traps (NETs). Furthermore, our data indicated that NETs inhibit Atox1 expression and leads to impaired ATP7A related copper transport and copper overload in cardiac microvascular endothelial cells (CMECs). Copper overload further leads to exacerbated cuproptosis, and these effects can be rescued by excision of the sympathetic nerve, targeted administration of copper chelating agent or NETs inhibitor. Conclusions: This study demonstrated that SF exacerbated MI/RI by promoting copper overload in CMECs. Our study elucidated one of the molecular mechanisms by which sleep disorders aggravate cardiac microvascular injury and suggested possible tar gets for intervention.