Objectives <p>To evaluate the association between apical periodontitis (AP) diagnosis and cytokine expression, exploring the mediating role of global 5-hydroxymethylcytosine (5-hmC) and the enzymes involved in DNA methylation and demethylation.</p> Materials and methods <p>A cross-sectional study included individuals with symptomatic AP (SAP; <i>n</i> = 18), asymptomatic AP (AAP; <i>n</i> = 19), and healthy periodontal ligament (HPL) (<i>n</i> = 11). mRNA levels of DNA methyltransferases (DNMT): DNMT1, DNMT3A; Ten-Eleven Translocation (TET) enzymes: TET2, TET3; and cytokines: IL-1β, and IL-6 were assessed by qRT-PCR in apical lesions and healthy periodontal ligament. Global %5-hmC was quantified by ELISA. Generalized structural equation models (GSEM) were used to evaluate the association between AP diagnosis and cytokine expression, considering %5-hmC and the expression of DNMT and TET genes as potential mediators, adjusted for age.</p> Results <p>SAP samples showed significantly higher DNMT1, DNMT3A, TET2, TET3, IL-1β, and IL-6 mRNA levels (<i>p</i> ≤ 0.05) and lower %5-hmC compared to HPL, but not when compared with AAP (<i>p</i> &gt; 0.05). In AAP and SAP, DNMT3A, TET2, and TET3 correlated positively with IL-1β and IL-6, while in SAP, DNMT1, DNMT3A, and TET3 were inversely correlated with %5-hmC (<i>p</i> ≤ 0.05), an association not observed in AAP (<i>p</i> &gt; 0.05). Furthermore, higher TET3 expression was associated with increased IL-1β and IL-6 mRNA levels (<i>p</i> ≤ 0.05) and with decreased global %5-hmC levels (<i>p</i> ≤ 0.05) in apical tissues.</p> Conclusions <p>Upregulated TET3 expression is associated with proinflammatory responses and global methylation profiles in apical lesions, suggesting a potential role in the epigenetic regulation of AP.</p> Clinical relevance <p>Epigenetic regulation of inflammatory cytokines may contribute to disease activity in periapical periodontitis.</p>