Background: Until now, no epigenome-wide association studies (EWAS) has investigated the impact of allergen immunotherapy (AIT) on DNA methylation in a longitudinal set-up. Herein, we investigated whether differences in DNA methylation occur in birch pollen allergic patients undergoing six months of birch pollen AIT, assessed alterations in methylation-based blood cell type composition, and correlated DNA methylation to serological AIT biomarkers. Methods: We performed genome-wide DNA-methylation analysis on bisulfite-converted DNA derived from whole blood samples of 16 birch pollen-allergic patients (pre-/post-birch pollen AIT) and 15 placebo (pre-/post-placebo treatment). Results: Our analysis identified cg22187251, located within a regulatory region upstream of the glucosaminyl (N-acetyl) transferase 2 ( GCNT2) gene and cg22336863 upstream of the transcription start site of actin binding rho activating protein ( ABRA), as differentially methylated. DNA methylation levels of cg22187251 in post-AIT-treated patients approximated those observed in a non-allergic reference cohort. Functional assays revealed that this region exhibits methylation-dependent promoter and enhancer activity. We identified differentially methylated sites within the HLA gene complex, and an AIT-specific increase of CD8+ T cell populations accompanied by a decrease in NK cell proportion. Moderate to strong correlations with clinical biomarkers (such as specific IgG ₄) were observed for 46% of the top 100 differentially methylated sites. Conclusions: GCNT2 and ABRA are implicated in Rho-signaling, a pathway involved in Th2 differentiation. GCNT2 modulates the SMAD-dependent TGF-β pathway, indicating a role in mediating AIT-induced immunotolerance. We provide evidence of DNA methylation within a regulatory region of a relevant gene, potentially restoring methylation levels to a non-allergic state.