Heparin has been documented to reduce myocardial injury caused by ischemia/reperfusion (I/R), but its clinical application is limited due to its strong intrinsic anticoagulant property. Some desulfated derivatives of heparin display low anticoagulant activity and may have potential value as therapeutic agents for myocardial I/R injury. In this study, we observed that 6-O-desulfated heparin, a desulfated derivative of heparin, shortened the activated partial thromboplastin time and exhibited lower anticoagulant activity compared with heparin or 2-O-desulfated heparin (another desulfated derivative of heparin). Then, we explored whether 6-O-desulfated heparin could protect against myocardial I/R injury, and elucidated its possible mechanisms. Administration of 6-O-desulfated heparin significantly reduced creatine kinase activity, myocardial infarct size and cell apoptosis in mice subjected to 30 min of myocardial ischemia following 2 h of reperfusion, accompanied by a reverse in miR-199a-5p elevation, klotho downregulation and reactive oxygen species (ROS) accumulation. In cultured H9c2 cells, the mechanism of 6-O-desulfated heparin against myocardial I/R injury was further explored. Consistent with the results in vivo, 6-O-desulfated heparin significantly ameliorated hypoxia/reoxygenation-induced injury, upregulated klotho and decreased miR-199a-5p levels and ROS accumulation, and these effects were reversed by miR-199a-5p mimics. In conclusion, these results suggested that 6-O-desulfated heparin with lower anticoagulant activity attenuated myocardial I/R injury through miR-199a-5p/klotho and ROS signaling. Our study may also indicate that 6-O-desulfated heparin, as an excellent heparin derivative, is a potential therapeutic agent for myocardial I/R injury.