Insulin resistance (IR) syndrome is associated with impaired vascular relaxation; however, the underlying pathophysiology is unknown. Potassium channel activation causes vascular smooth muscle hyperpolarization and relaxation. The present study determined whether a reduction in large conductance calcium- and voltage-activated potassium (BKCa) channel activity contributes to impaired vascular relaxation in IR rats. BKCachannels were characterized in mesenteric microvessels from IR and control rats. Macroscopic current density was reduced in myocytes from IR animals compared with controls. In addition, inhibition of BKCachannels with tetraethylammonium (1 mM) or iberiotoxin (100 nM) was greater in myocytes from control (70%) compared with IR animals (∼20%). Furthermore, activation of BKCachannels with NS-1619 was three times more effective at increasing outward current in cells from control versus IR animals. Single channel and Western blot analysis of BKCachannels revealed similar conductance, amplitude, voltage sensitivity, Ca2+sensitivity, and expression density between the two groups. These data provide the first direct evidence that microvascular potassium currents are reduced in IR and suggest a molecular mechanism that could account for impaired vascular relaxation in IR.