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Extending previous research on the genetic underpinnings of entrepreneurship, we investigate gender differences in the genetic influences on the tendency of people to become entrepreneurs. We also examined two mediating variables through which genetic factors may impact this tendency: extraversion and neuroticism. Based on 1285 pairs of identical twins (449 male and 836 female pairs) and 849 pairs of same-sex fraternal twins (283 male and 566 female pairs), we found that females have a strong genetic influence and zero shared-environmental influences on their tendency to become entrepreneurs. In contrast, males show zero genetic influence, but a large shared-environmental influence. Extraversion and neuroticism mediate the genetic influences on women's tendency to become entrepreneurs, whereas extraversion mediates shared-environmental influences on men's tendency to become entrepreneurs. We discuss this sharp difference in genetic influences on entrepreneurship across gender groups and highlight the different challenges that men and women face in their entrepreneurial endeavors.