Although high baseline testosterone levels correlate with low empathy, there is no causal evidence for this association in humans. The present study tested the causality of this relationship by manipulating testosterone levels in a double-blind placebo controlled crossover design. 20 healthy female participants received either a sublingual administration of a single dose of testosterone or placebo on 2 days and were tested 4 h after administration. Because research has shown that facial expression mimicry is a non-obtrusive index of empathy, facial electromyography was measured in response to dynamic facial expressions of happy and angry faces. Results showed that testosterone generally decreased facial mimicry. These findings are consistent with models that assign a critical role to mimicry in the ability to develop and communicate empathy towards conspecifics, and provide a potential causal mechanism of effects of testosterone on empathy.