Does testosterone increase the tendency to engage in self-employment? The results presented to date have been mixed. Using three different studies, we provide additional evidence on the relationship between testosterone and self-employment. Drawing on a cross section of 2,146 individuals (1,178 males and 968 females) from the National Health and Nutrition Examination Surveys’ 2011–2012 sample, and controlling for endogeneity (with red blood cell count, percentage hematocrit, and zinc supplement intake in the past 30 days as instruments), we find that serum testosterone levels are positively associated with self-employment for males (marginally significant, two-tailed test). As testosterone levels could be affected by social, economic, and biological factors during one’s life course, we draw more robust inferences by assessing whether the 2D:4D digit ratio, a marker of prenatal testosterone exposure, influences the likelihood of self-employment. From Understanding Society’s Innovation Panel Wave 6, we tested separate models for 449 males and 525 females, and our results indicate that males (respectively, females) with a lower 2D:4D ratio in their left hand, or higher prenatal testosterone exposure, have a significantly greater (respectively, marginally significant) likelihood of self-employment (two-tailed test). Finally, we examine the twin testosterone transfer effect in a sample of opposite-sex and same-sex twins from the National Survey of Midlife Development in the United States and provide additional support for the marginally significant (two-tailed test) positive association between testosterone and self-employment.Data are available at https://doi.org/10.1287/mnsc.2016.2664 .This paper was accepted by Toby Stuart, entrepreneurship and innovation.