Background: We evaluated the associations between 25-hydroxyvitamin D (25(OH)D) concentrations in serum and insulin resistance in the healthy Korean population. Methods: We conducted this cross-sectional analysis in 1,807 healthy Korean people (628 men and 1,179 women) aged 30 to 64 years in the Cardiovascular and Metabolic Disease Etiologic Research Center study. All participants were assessed for 25(OH)D, fasting glucose, and insulin levels, and completed a health examination and lifestyle questionnaire according to standard procedures. Insulin resistance was defined as the homeostasis model assessment insulin resistance higher than the 75 percentile. Results: Compared to those in the highest tertile (≥14.3 ng/mL), the odds ratio (OR) for insulin resistance was 1.37 (95% confidence interval [CI], 1.01 to 1.86) for the 1st tertile (<9.7 ng/mL) and 1.19 (95% CI, 0.08 to 1.62) for the 2nd tertile (9.7 to 14.3 ng/ mL) after adjusting for age, gender, waist circumference, alcohol consumption, smoking status, physical exercise, season, and cohort. After stratification of the subjects by adiposity, these associations remained only in non-obese subjects (lowest tertile vs. highest tertile, multivariable OR, 1.64; 95% CI, 1.05 to 2.56). Conclusion: Serum 25(OH)D has an independent inverse association with insulin resistance in the healthy, non-obese Korean population, even among people with vitamin D insufficiency.