The serotonin transporter promoter region polymorphism (5-HTTLPR) has been implicated in stress regulation, with increased stress reactivity often being found in carriers of the low-expressing short (S) allele. Nevertheless, the role of the 5-HTTLPR in influencing parasympathetic stress reactivity, as indexed by Respiratory Sinus Arrhythmia (RSA), is still unknown. This study examined, for the first time, whether the 5-HTTLPR was associated with variations in RSA response to maternal separation in a sample of 69 healthy 5-year-old children. Preschoolers' RSA was measured during an age-adapted version of the Strange Situation Procedure (SSP). The 5-HTTLPR polymorphism was tested as a predictor of RSA dynamic response to the SSP through multilevel models. A significant interaction between 5-HTTLPR and SSP episodes was found. In particular, whereas a significant decrease in RSA levels was observed during the stranger episode in the whole sample, S allele carriers showed a significant decrease in RSA levels from the stranger episode to the first separation episode, followed by an increase for the rest of the procedure. Albeit preliminary, data support the view that the 5-HTTLPR may contribute to individual differences in RSA stress reactivity from preschool age.