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<b><i>Aims:</i></b> The nerve growth factor (NGF) and the vascular endothelial growth factor-A (VEGF-A) may be of importance for psychiatric diseases including substance use disorders. The aim of the study was to identify differences in the regulation of both neuropeptides via the DNA-methylation status of the promotor regions of NGF and VEGF-A in different forms of maintenance therapy for opioid dependence and the related stress regulation via the hypothalamic-pituitary-adrenal axis. <b><i>Methods:</i></b> We compared methylation levels of opioid-dependent patients receiving treatment with diamorphine (<i>n</i> = 28) or levomethadone (<i>n</i> = 54) and similar levels in a healthy control group (<i>n</i> = 72). <b><i>Results:</i></b> There was a significantly higher methylation of VEGF-A in opioid-maintained patients with levomethadone compared to that in the control group (estimated marginal means [EMM] [SE]): 0.036 [0.003] vs. 0.020 [0.003]; <i>p</i> &#x3c; 0.001). We performed a cluster analysis for NGF, splitting up the results in 4 clusters. We found significant changes in methylation rates of the opioid-maintained patients compared to the controls in cluster I ([EMM] [SE]: 0.064 [0.005] vs. 0.084 [0.006]; <i>p</i> = 0.03), cluster II ([EMM] [SE]: 0.133 [0.013] vs. 0.187 [0.014]; <i>p</i> &#x3c; 0.001) and cluster III ([EMM] [SE]: 0.190 [0.014] vs. 0.128 [0.016]; <i>p</i> &#x3c; 0.001). <b><i>Conclusions:</i></b> The results are of importance, as they indicate that long-term changes in stress regulation regulated by neurotrophines are a crucial part of the symptomatology of opioid dependence, thus influencing drug consumption and the different forms of opioid-maintenance therapies.