Chrysosplenol C (4',5,6-trihydroxy-3,3',7-trimethoxyflavone) is a flavone contained in several medicinal plants including Miliusa balansae and Pterocaulon sphacelatum. This compound is known to have an antiviral effect and show cytotoxic activity in several cell lines. In the present study, we explored the effect of chrysosplenol C on contractility in isolated adult rat ventricular myocytes. Chrysosplenol C was isolated from M. balansae, and cell shortenings were measured in field-stimulated single myocytes using a video edge detection method at room temperature. Chrysosplenol C was found to increase cell shortenings in a dose-dependent manner with a half-maximal effective concentration of 45 ± 7.8 μM. Maximal effect of chrysosplenol C, approximately 185% of control, was observed at ≥80 μM. The positive inotropic effect caused by chrysosplenol C was reversible. Time-to-peak contraction and time-to-relengthening were significantly increased by chrysosplenol C. The velocity of cell shortening was slightly accelerated, whereas that of relaxation was not altered by chrysosplenol C. The chrysosplenol C–induced positive inotropic effect was not inhibited by propranolol posttreatment or H-89 pretreatment, suggesting that chrysosplenol C increased contraction independently of β-adrenergic receptor stimulation and protein kinase A. Our findings are the first to demonstrate that chrysosplenol C is a positive inotropic agent in cardiac myocytes.