Cardiovascular diseases show significant sex differences, which has been attributed to the cardioprotective effects of estrogen. Recent studies unravel that the underlying mechanisms seem unlikely to be due to estrogen only. There is a great variability in size of these vessels in females throughout life, suggesting that there is little contribution of sex hormones. To understand the mechanisms, the difference in coronary blood vessels size in the fetal murine heart was analyzed. A newly-developed coronary arteriogram enabled us to visualize the morphology of the coronary vessels in the murine heart at embryonic day 17.5. Although no sex difference was found in averaged length of left ventricle, we found that the hearts of both sexes were divided into two groups according to their size. Thus, diameter of left coronary vessel (LCV) was measured for each group of heart sizes and compared between males and females. We found a sex difference in the larger group that male LCVs were thicker than female LCVs. Administration of a NO-donor, NOC7, reversed the sex difference. Furthermore, our microarray analysis identified 59 genes with sex differences. These results indicate that sex differences in functional morphology of LCVs exist at a late embryonic stage in mice.