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 75 min balloon left anterior descending coronary occlusion and 3 h reperfusion. ODSH effects on [Na⁺]_i (NaGreen) and [Ca²⁺]I (Fluo-3) were measured by flow cytometry in rabbit ventricular myocytes after 45 min simulated ischemia (metabolic inhibition with 2 mM cyanide, 0 glucose, 37° C, pacing at 0.5 Hz, P-MI). 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 P-MI. The NCX inhibitor KB-R7943 (10μM), or the late Na⁺ current (INa-L) inhibitor ranolazine (10μM) reduced [Ca²⁺]_i during P-MI 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 (ATXII), a selective activator of the late Na current, I_Na-L. ODSH directly stimulated NCX and reduced I_Na-L. These results suggest that in the intact heart ODSH reduces Na⁺ influx during early reperfusion, when I_Na-L is activated by a burst of reactive oxygen production. This reduces Na⁺ overload and thus Ca²⁺ influx via Na⁺/Ca²⁺ exchange. Stimulation of Ca²⁺ extrusion via NCX later after reperfusion may also reduce myocyte Ca²⁺ loading and decrease infarct size.