Abstract Background Previous research reported that individual differences in the stress response were moderated by an interaction between individuals’ life stress experience and the serotonin transporter-linked polymorphic region (5-HTTLPR), a common polymorphism located in the promoter region of the serotonin transporter gene ( SLC6A4 ). Furthermore, this work suggested that individual differences in SLC6A4 DNA methylation could be one underlying mechanism by which stressful life events might regulate gene expression. The aim of this study was to understand the relation between early and recent life stress experiences, 5-HTTLPR genotype, and SLC6A4 methylation. In addition, we aimed to address how these factors influence gene expression and cortisol response to an acute psychosocial stressor, operationalized as the Trier Social Stress Test (TSST). In a sample of 105 Caucasian males, we collected early and recent life stress measures and blood samples to determine 5-HTTLPR genotype and SLC6A4 methylation. Furthermore, 71 of these participants provided blood and saliva samples before and after the TSST to measure changes in SLC6A4 and NR3C1 gene expression and cortisol response. Results Compared to S-group individuals, LL individuals responded with increased SLC6A4 mRNA levels to the TSST ( t (66) = 3.71, P