The purposes of this study were to investigate the effects of acute exercise on blood vaspin, glucose level, and inflammatory cytokines in middle-aged women with pre-diabetes. Nineteen middle-aged women (normal women, , n = 11) and women with pre-diabetes, (n = 8), who were apparently healthy and not taking any medications affecting their blood pressure, or blood glucose, participated. Body fatness parameters including body mass index, body fat percentage, and waist circumference were measured using a bio-impedance analyzer. Resting blood pressures was measured in duplicate, and mean values were used for the data analysis. Regardless of group assignments, all subjects participated in a 3-day consecutive walking exercise at an intensity of 65% VO2max targeting an energy expenditure of 1200 kcal (400 kcal per day). The major outcome variables included total cholesterol, triglycerides, high-density lipoprotein cholesterol, glucose, vaspin, interleukin-6, and adiponectin levels. Unlike normal women, women with pre-diabetes had a significant improvement in the homeostatic model assessment of insulin resistance (p < 0.025) with no significant group difference in response to acute exercise. The findings suggest that acute exercise results in a significant improvement in insulin sensitivity without any change in serum vaspin levels in women with pre-diabetes.