Tinctormine (1a), a novel N-containing quinochalcone C-glycoside, was isolated from the 60% acetone extract of dried flower petals of Carthamus tinctorius L. (Compositae) along with known compounds carthamin and safflor yellow B. Its structure was determined by means of 2-D NMR spectroscopy including HMBC, high-resolution FAB-MS, acetylation, and NOE experiments. The electrophysiological screening using a whole-cell voltage-clamp method on single canine ventricular myocytes revealed that tinctormine (1a, 10⁻⁵M) selectively inhibited the slow inward Ca²⁺ currents by approximately 42% of the control in a dose-dependent and reversible manner, without affecting the activation threshold (-40 mV) or reversal potential (+70 mV) of the Ca²⁺ currents. The inhibitory activity of 1a on Ca²⁺ currents was close to that of diltiazem (IC50: 5×10⁻⁶M) used in this preparation. Tinctormine (1a) was proved to be a potent Ca²⁺ antagonist and represents the first naturally occurring quinochalcone-type Ca²⁺ antagonist from C. tinctorius.