Morality is defined as prescriptive norms regarding how people should treat one another, and includes concepts of fairness, justice, and rights. One recent study with moral dilemmas suggested that testosterone administration increases utilitarian judgments, which depends on second-to-fourth (2D: 4D) digit ratio, as a proxy of prenatal priming. However, the neural mechanism by which acute testosterone modulates moral reasoning remains to be determined. Using a placebo-controlled within-subject design, the current study examined the neuromodulatory effect of testosterone in young females by combining moral dilemmas, 2D: 4D, functional magnetic resonance imaging (fMRI), and subjective ratings of morally laden scenarios. Results showed that testosterone administration elicited more utilitarian responses to evitable dilemmas. The high 2D: 4D group scored more punishments for moral evaluation, whereas the low 2D: 4D group did the opposite. The activity in the amygdala, anterior insular cortex, and dorsolateral prefrontal cortex (dlPFC) was increased when participants evaluated morally unorthodox actions (intentional harm). The activity in the posterior superior temporal sulcus/temporoparietal junction (pSTS/TPJ) to accidental harm was decreased, specific to the high 2D: 4D group. The functional connectivity between the amygdala and dlPFC was reduced. The activity in the pSTS/TPJ to perceived agency predicted utilitarian responses to evitable dilemmas. The findings demonstrate the acute effect of testosterone on neural responses associated with moral judgment, and provide evidence to support that prenatal sex-hormones priming could be important for early neurodevelopment, which plays a crucial role in the neural and behavioral manifestations of testosterone on adult moral reasoning. Hum Brain Mapp 37:3417-3430, 2016. © 2016 Wiley Periodicals, Inc.