Glyburide, also known as glibenclamide, was shown to have positive inotropic effect in human and animal hearts. The objectives of the present study was to investigate the effects of glyburide on developed left ventricular pressure (DLVP), coronary flow (CF), and heart rate (HR), in isolated rabbit heart as well as its effects on myocardial contractility and L-type calcium current, iCa, in guinea pig myocytes. Rabbit hearts were mounted on Langendorff apparatus and perfused with an oxygenated Krebs for 30 min until reaching steady state to be followed by 20 min of experimental perfusion divided into 5 min of control perfusion and 15 min of perfusion with Glyburide (10 μM). Ventricular myocytes were isolated by enzymatic dispersion technique and superfused in an oxygenated Tyrode solution. Cells were voltage-clamped at holding potential −40 mV to inactivate Na+ current and a step depolarizations, 200 msec duration, to 0 mV was applied to elicit iCa. The contractions of the myocytes were measured by optical methods. Glyburide significantly increased DLVP by 30% and CF by 36% but had no effect on HR. Glyburide increased cell contractility by 7 ± 6, 18 ± 7, 28 ± 9 and 54 ± 15% for 0.1, 1, 10 and 100 μM respectively, p < 0.001. Meanwhile it depressed iCa by 9 ± 6 and 19 ± 8% for 1 and 10 μM respectively. In conclusion, glyburide increased contractility of guinea pig single myocytes and of isolated rabbit heart, as indicated by increased developed left ventricular pressure while it depressed iCa. It is hypothesized that an elevation in intracellular calcium, which caused increased myocardial contractility, could be attributed to an increase in intracellular Na+ that could increase intracellular calcium via Na+/Ca2+ exchange.