Aim To explore the direct mechanism of melatonin (MT) on the large-conductance calcium-activated potassium channel (BKCa channel) in its inducing relaxation of mesenteric arteries. Methods Eight-week-old male Wistar rats were selected, the mesenteric arteries were removed. The mesenteric artery tension was measured by microvessel tension test, and the gating characteristics of BKCa channel in vascular smooth muscle cells were examined by single channel patch clamp technique. Results (1) MT had no significant effect on vascular tension, but it caused dose-dependent vascular relaxation in mesenteric arteries which were induced by norepinephrine (NE). The relaxation could be inhibited by luzindole (Luz) and iberiotoxin(IbTX), and the inhibition of IbTX was more intense. (2) The results of cell-attached patch clamp recordings revealed that MT significantly increased the open probability of BKCa channel and the average opening time, significantly reduced the average closing time; Luz significantly inhibited the activation of BKCa channel. (3)The results of inside-out patch clamp recordings showed that MT increased the open probability and the average opening time of BKCa channel and decreased the average closing time significantly. Conclusion MT activates the BKCa channels of smooth muscle cells in mesenteric arterier directly or indirectly through the receptor, which may be one of the important mechanisms underlying the inducing of MT on mesenteric arteries.