Human neutrophil peptides 1-3 (HNP1-3) contribute to oral health by playing a role in innate response against oral diseases, owing to their antimicrobial, antiplaque and immunomodulatory activity. Vitamin D may reduce the risk of infection through multiple mechanisms and boosts innate immunity by modulating the production of antimicrobial peptides and cytokine response. This study was performed to evaluate the level of human HNP1-3 in periodontitis patients and controls and to study the effect of vitamin D on the levels of peptides in periodontitis. Eighty-five subjects were enrolled in this observational case-control study aged 20-59 years. The subjects were divided into fifty patients with periodontitis and 35 healthy controls. Periodontal parameters used in this study were plaque index, gingival index bleeding on probing, probing pocket depth and clinical attachment loss. Blood samples were collected from all subjects, and then ELISA was done to estimate the levels of HNP1-3 and vitamin D in patients and controls. The finding revealed a significant increase (P˂0.01) in the mean level of HNP1-3 among the patient's group compared to the control group. Moreover, there is no significant correlation with all parameters except the gingival index, which has a considerable result (r=0.367, p=0.009). On the other hand, this study showed a significant decrease (P<0.01) in the mean level of vitamin D in patients compared to controls, and also that there was no significant decrease in the serum level of HNP1-3 among vitamin D insufficient group in comparison to vitamin D sufficient group. These results indicated that the increased levels of HNP1-3 and its correlation with GI provide evidence for the critical role of neutrophils in the inflammatory process in periodontitis and vitamin D, which plays a role in antimicrobial peptide production. Keywords: periodontitis, antimicrobial peptides, human neutrophils peptides