It has been well documented that matrine, a tetracyclo-quinolizindine alkaloid, possessed a positive inotropic effect. However, the underlying mechanisms at the cellular and ion channel levels have not been completely clarified. Therefore, the present study was designed to identify the cellular target and the mechanisms of inotropic effect of matrine. Guinea pig papillary muscles were used to study the contractile force of the heart and ventricular myocytes were used to study L-type calcium channel (ICa-L) and intracellular calcium concentration ([Ca2+]i). In electrically driven papillary muscles, matrine enhanced the contractile force in a dose-dependent manner and the positive inotropic effect was not inhibited by alpha- and beta-adrenergic receptor antagonists. In ventricular myocytes, matrine also increased ICa-L in a dose-dependent manner and shifted the inactivation curve toward right. Matrine markedly enhanced the KCl-induced elevations of [Ca2+]i. In a conclusion, ICa-L might be a main target of matrine. Matrine enhanced [Ca2+]i by stimulating ICa-L and exerted positive inotropic effects on electrically driven guinea pig papillary muscles.