Alpha₁-adrenoceptor stimulation (α₁ARS) shows a positive inotropic effect in most mammalian myocardium. In mouse myocardium, however, α₁ARS showed the negative inotropic effect, of which intracellular mechanisms are not fully clarified. The purpose of this study is to investigate the intracellular mechanism of the negative inotropic effect by α₁ARS in C57BL/6 mouse myocardium. We used isolated ventricular papillary muscles of C57BL/6 strain mouse which is widely used for genetic manipulation. We simultaneously measured isometric tension and intracellular Ca²⁺ concentration ([Ca²⁺]_i) using the aequorin method. In twitch contraction, phenylephrine concentration-dependently (1–100 μM) decreased tension without significant changes in the Ca²⁺ transient, and these effects were completely blocked by prazosin (3 μM) or calphostin C (a PKC inhibitor, 1 μM). Phorbol 12-myristate 13-acetate (PMA) (a PKC activator, 1 μM) decreased tension as observed in phenylephrine. After PMA application, the negative inotropic effect of phenylephrine disappeared. To estimate the Ca²⁺ sensitivity, tetanic contraction was produced, and the relation between [Ca²⁺]_i and tension at a steady state was measured. Phenylephrine (10 μM) decreased the Ca²⁺ sensitivity, and PMA showed a similar Ca²⁺ desensitizing effect. These results suggest that the negative inotropic effect of phenylephrine in mouse myocardium can be explained by the decrease in the Ca²⁺ sensitivity through the activation of PKC. The present result indicates that the effect of α₁ARS differs among species and strains of experiment animals. Thus, we should be careful about using the results of mouse myocardium to understand the functions of the human heart.