Males and females score differently on some personality traits, but the underlying etiology of these differences is not well understood. This study examined genetic, environmental, and prenatal hormonal influences on individual differences in personality masculinity–femininity (M-F). We used Big-Five personality inventory data of 9,520 Swedish twins (aged 27 to 54) to create a bipolar M-F personality scale. Using biometrical twin modeling, we estimated the influence of genetic and environmental factors on individual differences in a M-F personality score. Furthermore, we tested whether prenatal hormone transfer may influence individuals’ M-F scores by comparing the scores of twins with a same-sex versus those with an opposite-sex co-twin. On average, males scored 1.09 standard deviations higher than females on the created M-F scale. Around a third of the variation in M-F personality score was attributable to genetic factors, while family environmental factors had no influence. Males and females from opposite-sex pairs scored significantly more masculine (both approximately 0.1 SD) than those from same-sex pairs. In conclusion, genetic influences explain part of the individual differences in personality M-F, and hormone transfer from the male to the female twin during pregnancy may increase the level of masculinization in females. Additional well-powered studies are needed to clarify this association and determine the underlying mechanisms in both sexes.