Weaning, particularly the widespread practice of abrupt separation of the mare and foal, has been shown to be a stressful event for horses. Physiological changes in foals measured after weaning include increased blood cortisol concentrations and a subsequent decrease in cell-mediated immune responses. In the randomized, double-blinded, placebo-controlled trial reported here, we assessed the effect of an equine appeasing pheromone (EAP; Modipher EQ, E.A.P. Mist [Pherosynthese s.n.c., Le Rieu Neuf, Saint-Saturnin-les-Apt, France]) as an aid for reducing the behavioral and physiological signs of stress during weaning. Fourteen quarter horse foals were separated from their dam (equid mother) between 105 and 146 days of age, in age-matched pairs, and placed in 3.66 × 3.66 m stalls (one treated and one control foal in each stall). Treated foals received the synthetic analogue of the EAP by intranasal wipe 30 minutes before separation and twice daily thereafter for 48 hours. Control foals received placebo by intranasal wipe on the same schedule. The foals were continuously videotaped for 48 hours postweaning to monitor behavioral responses. Blood samples were drawn 24 hours pretreatment and 24 and 48 hours postweaning for evaluation of physiological indicators of stress (serum cortisol) and cytokines as stress-related and immune-mediated response parameters. Interestingly, although behavioral and serum cortisol measures were similar between groups, treatment with EAP had a significant (P < 0.05) effect on interleukin 6 and transforming growth factor β, whereas monocyte opiate receptor 1 was significantly upregulated in both groups independent of treatment when compared with baseline values. Although the link between EAP treatment and activation of the measured cytokines remains unexplained, our findings suggest immune-related gene transcription focused on the acute causes of stress in a time frame shortly after weaning.