ObjectiveThe aim of the study was to assess whether or not Vitamin D deficiency was associated with the GCF and gingival tissue antimicrobial peptides (AMP), namely, human beta defensin-2 (hBD-2) and cathelicidin (LL-37) level in chronic periodontitis (CP) and gingivitis patients. DesignA total of 80 volunteers were included in this study. Forty was classified as Vitamin D deficient (25(OH)D <20 ng/mL), and 40 Vitamin D sufficient patients (25(OH)D ≥ 20 ng/mL). Of these, 20 of them were affected by gingivitis and 20 by CP. Following sampling, the hBD-2 and LL-37 concentration in gingival tissues and GCF were determined by the ELISA method. ResultsThe hBD-2 and LL-37 levels were higher in periodontitis compared to gingivitis patients within Vitamin D sufficient and deficient groups. The AMP levels of GCF and gingival tissue in the vitamin D deficient group was lower compared to sufficient serum 25(OH)D within gingivitis and CP groups. Additionally, a non-parametric regression model known as the generalized additive model was used to identify the contribution of diagnosis, Vitamin D status, and other potential clinical variables on the local levels of AMPs. Regression analysis showed that the periodontal disease status, serum vitamin D concentration were independent predictors for elevated GCF AMP levels. Finally, a positive correlation between GCF and tissue levels of both hBD-2 (r = 0.82; <0.0001) and LL-37 (r = 0.65; <0.0001) was detected. ConclusionThis study shows that serum 25(OH)D deficiency is associated with decreased hBD-2 and LL-37 expression of GCF and gingival tissues in both gingivitis and CP patients.