Aim The presence of Gram‐negative anaerobic bacteria, in particular, Porphyromonas gingivalis (P. gingivalis) in periapical granulomas predicts the generation of citrullinated proteins in the lesion. Citrullination of proteins may lead to the formation of anti‐citrullinated autoantibodies (ACPA‐s) initiating the formation of an autoimmune loop which may contribute to the perpetuation of inflammatory reactions and tissue damage in chronic apical periodontitis. The objective of this study was to demonstrate the formation of citrullinated proteins in chronic apical periodontitis and whether they can act as autoantigens. Methodology Twenty‐five periapical granulomas (n = 25) were investigated in the study. Healthy periodontal tissue samples served as normal control tissue (n = 6). The peptidyl‐citrulline level was determined with the dot blot method. ACPA levels were analysed using anti‐citrullinated cyclic peptide (anti‐CCP) EDIA kit. Differences between periapical granuloma and control samples were assessed using Mann–Whitney U tests. p Values <.05 were considered as statistically significant. Results Protein concentrations, peptidyl‐citrulline levels and anti‐CCP ratios were compared between periapical granuloma and healthy control groups. Multiple linear regression analysis revealed significant (p = .042) hypercitrullination in periapical granuloma samples. Moreover, there was a significant difference in the ACPA ratios between periapical granuloma (2.03 ± 0.30) and healthy control (0.63 ± 0.17) groups (p = .01). Seventeen of 25 periapical granuloma samples (17/25; 68%), whereas one out of six control samples (1/6; 17%) were shown to be positive for the presence of ACPA. Conclusions This is the first study detecting the presence of citrullinated peptides and APCA in periapical granuloma, suggesting the contribution of autoimmune reactions in the pathogenesis and perpetuation of chronic apical periodontitis.