Testosterone is an important hormone in the sexual differentiation of the brain, contributing to differences in cognitive abilities between males and females. For instance, studies in clinical populations such as females with congenital adrenal hyperplasia (CAH) who are exposed to high levels of androgens in utero support arguments for prenatal testosterone effects on characteristics such as visuospatial cognition and behaviour. The comparison of opposite-sex (OS) and same-sex (SS) twin pairs can be used to help establish the role of prenatal testosterone. However, although some twin studies confirm a masculinizing effect of a male co-twin regarding for instance perception and cognition it remains unclear whether intra-uterine hormone transfer exists in humans. Our aim was to test the potential influences of testosterone on academic performance in OS twins. We compared ninth-grade test scores and teacher ratings of OS (n = 1812) and SS (n = 4054) twins as well as of twins and singletons (n = 13,900) in mathematics, physics/chemistry, Danish, and English. We found that males had significantly higher test scores in mathematics than females (.06–.15 SD), whereas females performed better in Danish (.33–.49 SD), English (.20 SD), and neatness (.45–.64 SD). However, we did not find that OS females performed better in mathematics than SS and singleton females, nor did they perform worse either in Danish or English. Scores for OS and SS males were similar in all topics. In conclusion, this study did not provide evidence for a masculinization of female twins with male co-twins with regard to academic performance in adolescence.