In addition to increased glucose uptake, insulin action is associated with increased total and microvascular blood flow, and vasomotion in skeletal muscle. The aim of this study was to determine the effect of acute insulin resistance caused by the peripheral vasoconstrictor α-methylserotonin (αMT) on microvascular vasomotion in muscle. Heart rate (HR), mean arterial pressure (MAP), femoral blood flow (FBF), whole body glucose infusion (GIR) and hindleg glucose uptake (HGU) were determined during control and hyperinsulinaemic euglycaemic clamp conditions in anaesthetized rats receiving αMT infusion. Changes in muscle microvascular perfusion were measured by laser Doppler flowmetry (LDF) and vasomotion was assessed by applying wavelet analysis to the LDF signal. Insulin increased GIR and HGU. Five frequency bands corresponding to cardiac, respiratory, myogenic, neurogenic and endothelial activities were detected in the LDF signal. Insulin infusion alone increased FBF (1.18 ± 0.10 to 1.78 ± 0.12 ml min-1, P < 0.05), LDF signal strength (by 16% compared to baseline) and the relative amplitude of the myogenic component of vasomotion (0.89 ± 0.09 to 1.18 ± 0.06, P < 0.05). When infused alone αMT decreased LDF signal strength and the myogenic component of vasomotion by 23% and 27% respectively compared to baseline, but did not affect HGU or FBF. Infusion of αMT during the insulin clamp decreased the stimulatory effects of insulin on GIR, HGU, FBF and LDF signal and blocked the myogenic component of vasomotion. These data suggest that insulin action to recruit microvascular flow may in part involve action on the vascular smooth muscle to increase vasomotion in skeletal muscle to thereby enhance perfusion and glucose uptake. These processes are impaired with this model of αMT-induced acute insulin resistance.