Sex differences in endothelial cell (EC) biology may reflect intrinsic differences driven by chromosomes or sex steroid exposure and gender differences accumulated over life. We analysed EC gene expression data from boy-girl twins at birth and in non-twin adults to detect sex differences at different stages of life, and show that 14-25% of the EC transcriptome is sex-biased. By combining data from both stages of life, we identified sex differences that are present at birth and maintained throughout life, and those that are acquired over life. Promisingly, we found that genes that present with an acquired sex difference in ECs are more likely to be targets of sex steroids. Annotating both gene sets with data from multiple genome-wide association studies (GWAS) revealed that genes with an intrinsic sex difference in ECs are enriched for coronary artery disease GWAS hits. This study underscores the need for treating sex as a biological variable.