To investigate the relationship between serum high-density lipoprotein (HDL-C) and spinal bone mineral density (BMD) under different serum 25-hydroxyvitamin D (25 (OH) D) levels in adults over 40 years old and to explore its mechanism. We include participants over the age of 40 with data on HDL-C, 25 (OH) D, spinal BMD, and other variables in the National Health and Nutrition Examination Survey 2007-2010 in the analysis. A weighted multiple linear regression model was used to evaluate the association between serum HDL-C and spinal BMD in different gender, ages, and serum 25 (OH) D levels. A total of 3599 subjects aged ≥ 40 years old were included in this study. Univariate analysis of the complete correction model showed a negative correlation between serum HDL-C and spinal BMD. In the two subgroups of serum 25 (OH) D, we found that the higher the serum HDL-C in the female with serum 25 (OH) D < 75 nmol/L aged 40-59 years old, the lower the total spinal BMD, and a similar relationship was found in the lumbar spine. However, no similar relationship was found in all populations with serum 25 (OH) D ≥ 75 nmol/L and males with serum 25 (OH) D < 75 nmol/L. These results suggest that among Americans over the age of 40, the increase in serum HDL-C is related to decreased BMD of spine only in women aged 40-59 years with vitamin D insufficiency or deficiency.© 2023. The Author(s).