The hypothalamic-pituitary-adrenal (HPA) axis plays a major role in the development, elicitation, and enhancement of aggressive behavior in animals. Increasing evidence suggests that this is also true for humans. Here, we report on a study of the role of basal and acute HPA axis activity in a sample of 48 healthy male and female adults. We pharmacologically enhanced cortisol levels and used the Taylor Aggression Paradigm (TAP) to induce and measure aggression (divided into three blocks). Participants either received an oral dose of 20 mg hydrocortisone (cortisol group) or a placebo (placebo group). Half of each group received high or low levels of provocation with the TAP, respectively. Before, we assessed the cortisol awakening response as a trait measure of basal HPA axis activity. Participants in the cortisol group reacted more aggressively in the third block of the TAP compared to the placebo group. Furthermore, gender interacted with treatment: only females, but not males showed enhanced aggressive behavior after cortisol administration. There was no significant difference in males between the placebo and cortisol group. Basal HPA axis activity was negatively related to aggressive behavior, but again only in females and most strongly within the placebo group. This study provides the first evidence for a causal involvement of acute HPA axis activation in aggressive behavior in humans.