Background The glucocorticoid receptor (GR) 1F region is involved in transcription and expression of the GR protein and influences hypothalamic-pituitary-adrenal (HPA)-axis activity. Several studies have investigated GR-1F DNA methylation in the context of traumatic stress and psychiatric disorders, such as major depressive disorder and posttraumatic stress disorder (PTSD). However, longitudinal studies examining GR-1F DNA methylation before and after exposure to traumatic stress are lacking. We therefore aimed to investigate prospective DNA methylation changes in the GR-1F region after military deployment and its relation to the emergence of psychopathology. Methods Whole blood DNA methylation in the entire GR-1F region (52 CpGs) before and six months after deployment was quantified using pyrosequencing (N=92). Methylation levels were linked to post-deployment mental health problems (Revised Symptom Checklist, SCL-90), PTSD symptoms (Self-Rating Inventory for PTSD) and trauma exposure during deployment. Moreover, methylation was related to GR-1F expression, GR binding and genetic variation in the GR. Mean methylation, the number of methylated sites (methylation burden), mean methylation at transcription factor binding sites and at CpGs significantly associated with GR-1F expression (functional methylation) were examined. Results Trauma exposure during deployment and the emergence of mental health problems were significantly related to an increased methylation burden (t=2.23, p=2.8x10-2, =0.45 and t=2.24, p=2.7x10-2 and =0.085, respectively), which was associated with both decreased GR-1F expression (t=-2.92, p=4.7x10-3, =-1.93) and GR binding (t=-2.13, p=3.9x10-2, =-2.9x10-3). Moreover, development of psychopathology symptoms was significantly associated with increased methylation at functionally relevant CpGs (mental health problems: t=3.73, p=3.5x10-4, =1.2x10-2; PTSD symptoms: t=2.10, p=3.8x10-2, =0.59). Change in mean methylation was associated with a change in mental health problems (t=1.99, p=5.0x10-2, =4.1x10-3) and in GR-1F expression (t=-2.35, p=2.2x10-2, =-0.074). Discussion This longitudinal study in a military cohort shows that GR-1F DNA methylation levels are related to trauma exposure during deployment and the development of post-deployment psychopathology symptoms, particularly at functionally relevant sites. Together, our results provide further insight in transcriptional regulation of the glucocorticoid receptor gene, by demonstrating that GR-1F DNA methylation levels can vary over time and are related to stress vulnerability and the emergence of post-deployment psychopathology symptoms.