Abnormal serotonin transporter (5-HTT) function and autonomic nervous system (ANS) dysregulation has been proposed in panic disorder. However, in contrast to hypothalamo-pituitary-adrenocortical (HPA) functioning, ANS reactivity during panic response has yet not been investigated in humans with respect to the 5-HTT genotype. The present study assessed the influence of challenging by cholecystokinin tetrapeptide (CCK-4) on heart rate variability (HRV) measures, to monitor autonomic reactivity and its relationship to 5-HTT-linked polymorphic region (5-HTTLPR) genotypes. We hypothesized substantial effects of the 5-HTTLPR genotype on autonomic reactivity. We studied 30 healthy young men, 15 of each with the long/long (l/l) or short/short (s/s) genotype for the 5-HTTLPR. All participants received an intravenous application of 50 μg CCK-4. HRV measures were assessed in both groups at baseline and immediately after CCK-4 application. Our results indicated lower parasympathetic activity in s/s carriers during baseline, time and frequency domain measures. CCK-4 application significantly enhanced the sympathetic tone in both groups, leading to diminished group differences. A significant treatment by genotype effect indicated reduced autonomic reactivity to CCK-4 challenge in the s/s compared to l/l carriers. Our findings show enhanced sympathetic and/or diminished cardiac vagal activity under basal conditions and blunted autonomic reactivity in s/s vs. l/l carriers. Our study provides novel data supporting claims that the s/s genotype represents a genetic vulnerability factor associated with inadequate hyporeactivity to stress and extends current knowledge on the impact of the central serotonergic activity on the sympathoadrenal pathway.