Brazilein (6a,7-dihydro-3,6a,10-trihydroxy-benz[b]indeno[1,2-d]pyran-9(6H)-one) is a compound obtained in a large amount from Caesalpinia sappan ethanol extracts with a high purity of about 98%. In isolated cardiac tissues, we found that brazilein exhibited a positive inotropic action in a concentration-dependent manner with little effect on heart rate and coronary perfusion. To study its possible mode of action, isolated rat hearts were treated with propranolol. This treatment did not alter the cardiotonic effect of brazilein, suggesting that this effect does not involve stimulation of β-adrenoceptors. On the other hand, an analysis of the interaction between Na⁺,K⁺-ATPase and brazilein was carried out. Albino guinea pig erythrocytes (mainly α1-Na⁺,K⁺-ATPase isoforms) enriched with Na⁺,K⁺-ATPase isoforms were utilized to compare the inhibition promoted by brazilein with that of classical inhibitors such as the cardiac glycoside deslanoside. Analysis of inhibition curves revealed that unlike deslanoside, brazilein had a relatively low affinity for erythrocyte isoforms and failed to completely inhibit the Na⁺,K⁺-ATPase activity. The extent of the maximum inhibition rate was about 50%. The inhibitory effect of brazilein was not antagonized by 10 mmol/l K⁺, as observed with deslanoside. Electrocardiogram research in vivo showed that brazilein did not induce the ventricular arrhythmias observed with deslanoside, suggesting that brazilein might have a less adverse effect and higher therapeutic index than cardiac glycosides. In light of all the above-mentioned observations, it can be concluded that brazilein, a molecule with a non-steroidal skeleton, produced its positive inotropic effect through inhibiting Na⁺,K⁺-ATPase and could thus serve as a structural paradigm to develop new inotropic drugs.