In rodents stress impairs delay as well as trace eyelid conditioning in females, but enhances it in males. The present study tested the effects of acute psychosocial stress exposure on classical delay eyeblink conditioning in healthy men and women. In a between subject design, participants were exposed to psychosocial stress using the Trier Social Stress Test (TSST) or a control condition which was followed by a delay eyeblink classical conditioning procedure. Stress exposure led to a significant increase in salivary cortisol and impaired acquisition of conditioned eyeblink responses (CRs). This was evident by a later first CR and an overall lower CR rate of the stress group. The stress-induced acquisition impairment was observed in both women and men. Subjects failing to show a stress-induced cortisol increase (cortisol non-responder) were not impaired in acquisition. Our findings indicate that acute stress, possibly via activation of the hypothalamus-pituitary-adrenal (HPA) axis, reduces the ability to acquire a simple conditioned motor response in humans.