Bipolar Disorder (BD) is a prevalent and disabling condition, determined by gene-environment interactions, possibly mediated by epigenetic mechanisms. The present study aimed at investigating the transcriptional regulation of BD selected target genes by DNA methylation in peripheral blood mononuclear cells of patients with a DSM-5 diagnosis of type I (BD-I) and type II (BD-II) Bipolar Disorders (n = 99), as well as of healthy controls (CT, n = 42). The analysis of gene expression revealed prodynorphin (PDYN) mRNA levels significantly reduced in subjects with BD-II but not in those with BD-I, when compared to CT. Other target genes (i.e. catechol-O-methyltransferase (COMT), glutamate decarboxylase (GAD67), serotonin transporter (SERT) mRNA levels remained unaltered. Consistently, an increase in DNA methylation at PDYN gene promoter was observed in BD-II patients vs CT. After stratifying data on the basis of pharmacotherapy, patients on mood-stabilizers (i.e., lithium and anticonvulsants) were found to have lower DNA methylation at PDYN gene promoter. A significantly positive correlation in promoter DNA methylation was observed in all subjects between PDYN and brain derived neurotrophic factor (BDNF), whose methylation status had been previously found altered in BD. Moreover, among key genes relevant for DNA methylation establishment here analysed, an up-regulation of DNA Methyl Transferases 3b (DNMT3b) and of the methyl binding protein MeCP2 (methyl CpG binding protein 2) mRNA levels was also observed again just in BD-II subjects. A clear selective role of DNA methylation involvement in BD-II is shown here, further supporting a role for BDNF and its possible interaction with PDYN. These data might be relevant in the pathophysiology of BD, both in relation to BDNF and for the improvement of available treatments and development of novel ones that modulate epigenetic signatures.