Nitric Oxide (NO) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle cells (VSMCs). However, the role of NO in the electrophysiology of human artery SMCs is rarely studied. The aim of this study was to characterize the role of NO on BKCa channels in SMCs freshly isolated from human mesenteric arteries. The use of sodium nitroprusside (SNP) as NO donor in solutions was utilized. The freshly isolated SMCs were used to record BKCa currents using the whole-cell, amphotericin-perforated configuration of patch-clamp technique. Our data show that BKCa currents are often recorded as current spikes (STOCs) and macroscopic currents (MC) under whole-cell voltage clamp conditions. Human mesentery artery SMCs have an average current density of 2.92±0.355 pF/pA (at 0 mV), 16.02±1.15 (at +60 mV) (n=90). In human mesentery artery SMCs, STOCs existed random nature and varied widely from cell to cell and with time, as well as membrane potential. At -30 mV membrane potential, the amplitude was 6-60 pA and frequency 0.03-8 Hz, and at 0 mV, the amplitude (10-120 pA) and frequency (0.5-13 Hz). Extracellular application of SNP enhanced both MC and STOCs. 50 µM SNP increased MC by (8.3±2.3)% (at 0 mV), (29.5% ±16.8)% ( at +60mV) (n=3). 100 µM SNP increased MC by (34.6±30.)% (at 0 mV), (41.7±21.1)% ( at +60 mV) (n=9), and 200 µM increased MC by (30.9±26.7)% (at 0 mV), (45.5±16.0)% (at +60mV) (n=5). At -30 mV membrane potential, 100 µM SNP increased the frequency of STOCs from 3.32±0.85 (Hz) to 5.43 ±0.75 (Hz) (n=6, p<0.05) and amplitude of STOCs from 16.84±1.84 to 18.40±2.61 (pA) (n=6). 200 µM NO induced the frequency to 4.70±1.03 (Hz) (n=4) and amplitude to 16.81±3.15 (pA) (n=4). NO existed short-term effect on BKCa and its action often lasted no more than 10 min after each administration of SNP. The reversibility of NO on BKCa channels was investigated and was determined. Our results suggested that NO could stimulate BKCa channels of human mesenteric artery SMCs in a time dependent and reversible manner.