Heparin desulfated at the 2- O and 3- O positions (ODSH) decreases canine myocardial reperfusion injury. We hypothesized that this occurs from effects on ion channels rather than solely from anti-inflammatory activities, as previously proposed. We studied closed-chest pigs with balloon left anterior descending coronary artery occlusion (75-min) and reperfusion (3-h). ODSH effects on [Na+]i (Na Green) and [Ca2+]i (Fluo-3) were measured by flow cytometry in rabbit ventricular myocytes after 45-min of simulated ischemia [metabolic inhibition with 2 mM cyanide, 0 glucose, 37°C, pacing at 0.5 Hz; i.e., pacing-metabolic inhibition (PMI)]. Na+/Ca2+ exchange (NCX) activity and Na+ channel function were assessed by voltage clamping. ODSH (15 mg/kg) 5 min before reperfusion significantly decreased myocardial necrosis, but neutrophil influx into reperfused myocardium was not consistently reduced. ODSH (100 μg/ml) reduced [Na+]i and [Ca2+]i during PMI. The NCX inhibitor KB-R7943 (10 μM) or the late Na+ current ( INa-L) inhibitor ranolazine (10 μM) reduced [Ca2+]i during PMI and prevented effects of ODSH on Ca2+ loading. ODSH also reduced the increase in Na+ loading in paced myocytes caused by 10 nM sea anemone toxin II, a selective activator of INa-L. ODSH directly stimulated NCX and reduced INa-L. These results suggest that in the intact heart ODSH reduces Na+ influx during early reperfusion, when INa-L is activated by a burst of reactive oxygen production. This reduces Na+ overload and thus Ca2+ influx via NCX. Stimulation of Ca2+ extrusion via NCX later after reperfusion may also reduce myocyte Ca2+ loading and decrease infarct size.