Objective:  This study aimed to investigate the expression patterns of the newly discovered human β‐defensin‐3 (hBD‐3) in human gingiva.Background:  Human β‐defensins (hBDs) are a group of small, broad‐spectrum, cationic antimicrobial peptides. Our recent study showed that the expression levels of hBD‐1 and 2 peptides were associated with periodontal conditions.Methods:  A total of 49 gingival biopsies were collected, including 33 samples from 21 patients with chronic periodontitis and 16 samples from 16 periodontally healthy subjects. The expression of hBD‐3 was detected by immunohistochemistry and in situ hybridization. Double staining was undertaken to identify hBD‐3 peptide‐positive cells, using CD‐1a and cytokeratin 20 as markers for Langerhans cells and Merkel cells, respectively.Results:  hBD‐3 peptide was detected in 88% of the samples, which was confined to the gingival epithelia. In healthy control subjects, hBD‐3 peptide was more frequently detected in the basal layer as compared to the patients (53% vs. 18%, p < 0.05). In patients, hBD‐3 expression extended from the basal layer to the spinous layers (82%), in which hBD‐3 was confined to the basal and deep spinous layers in clinically healthy tissues from patients, whereas it extended to the superficial spinous layers in pocket tissues from patients (0% vs. 50%, p < 0.05). In both groups, hBD‐3 peptide was expressed not only in gingival keratinocytes, but also in Langerhans cells and Merkel cells. hBD‐3 transcripts were detected in 90% of the samples and they were confined to the basal and/or suprabasal layers of gingival epithelia.Conclusions:  This study shows that hBD‐3 is frequently expressed in gingival epithelia. The appropriate expression of hBD‐3 peptide may contribute to the maintenance of periodontal homeostasis, possibly through its antimicrobial effect and promotion of adaptive immune responses.