The aim of this study was to clarify if phentolamine has proven effects on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine involving the ATPsensitive $K^+$ channels and adrenergic receptor. The actions of phentolamine on pacemaker activities were investigated using whole-cell patch-clamp technique and intracellular $Ca^{2+}$ analysis at $30^{circ}C$ in cultured mouse intestinal ICC. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. Treatment with phentolamine reduced the frequency and amplitude of the pacemaker currents and increased the resting outward currents. Moreover, under current clamping (I = 0), phentolamine hyperpolarized the ICC membrane and decreased the amplitude of the pacemaker potentials. We also observed that phentolamine inhibited spontaneous $[Ca^{2+}]_i$ oscillations in ICC. The alpha-adrenergic drugs prazosin, yohimbine, methoxamine, and clonidine had no effect on ICC intestinal pacemaker activity and did not block phentolamine-induced effects. Phentolamine-induced effects on the pacemaker currents and the pacemaker potentials were significantly inhibited by ATP sensitive $K^+$ channel blocker glibenclamide, but not by TEA, apamin, or 4-aminopyridine. In addition, the NO synthase inhibitor, L-NAME and the guanylate cyclase inhibitor, ODQ were incapable of blocking the phentolamine-induced effects. These results demonstrate that phentolamine regulates the pacemaker activity of ICC via ATP-sensitive $K^+$ channel activation. Phentolamine could act through an adrenergic receptor- and also through NO-independent mechanism that involves intracellular $Ca^{2+}$ signaling.