Background: The objective of this study was to examine the cross-sectional relationship between homeostasis model assessment for insulin resistance (HOMA-IR) and serum 25-hydroxyvitamin D (25-OHD) level in Chinese children and adolescents. Methods: Anthropometric indices, lipid metabolic profile, and serum levels of glucose, insulin and 25-OHD were determined among 278 healthy prepubertal and pubertal, normal and overweight/obese children and adolescents aged 8-18 years between March 2014 and February 2015. Results: HOMA-IR was significantly different across vitamin D statuses (p < 0.001), even after adjusting for body mass index (BMI) (p = 0.035) and waist-to-height ratio (p = 0.044); the difference was not significant between the vitamin D deficient and insufficient groups (p = 0.120). HOMA-IR negatively correlated with serum 25-OHD level for all subjects (R-2 = 0.148, p < 0.001). Furthermore, they negatively correlated in the normal (R-2 = 0.160, p < 0.001) and overweight/obese (R-2 = 0.086, p < 0.001) groups, respectively. The regression lines of the two groups were parallel (p = 0.669) but had a significantly different intercept (p < 0.001). An association between HOMA-IR and BMI and serum 25-OHD level (R-2 = 0.654, p < 0.001) was demonstrated based on the stepwise multiple linear regression analysis of age, sex, pubertal maturation, BMI, waist-to-height ratio, triglyceride, cholesterol, high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), 25-OHD and HOMA-IR. Conclusions: Our findings supported that lower vitamin D status is strongly associated with worse HOMA-IR.