Mimicry, and especially spontaneous facial mimicry, is a rudimentary element of social-emotional experience that is well-conserved across numerous species. Although such mimicry is thought to be a relatively automatic process, research indicates that contextual factors can influence mimicry, especially in humans. Here, we extend this work by investigating the effect of acute psychosocial stress on affiliative facial mimicry. Participants performed a spontaneous facial mimicry task with facial electromyography (fEMG) at baseline and approximately 1-month later, following an acute psychosocial stressor (Trier Social Stress Test). Results show that the magnitude of the endocrine stress response reduced zygomaticus major reactivity, and specifically spontaneous facial mimicry for positive social stimuli (i.e., smiles): Individuals with higher levels of the stress hormone cortisol showed a more blunted fEMG response to smiles, but not to frowns. Conversely, stress had no effect on corrugator supercilii activation (i.e., frowning to frowns). These findings highlight the importance of the biological stress response system in this basic element of social-emotional experience.