The epidermis functions as a first-line defense barrier that protects the body from the external environment. As a chemical hindrance, the epidermis possesses acidic pH, highly organized lipids and various host defense peptides, also known as antimicrobial peptides. Human β-defensins (hBDs), one of the most important host defense peptide families found in our skin, are well-known for their broad-spectrum microbicidal activities. However, there is a growing body of evidence indicating that hBDs also orchestrate several immunomodulatory functions and are the cornerstone that bridges the innate and adaptive immune responses during skin inflammation and infection. Moreover, recent work identified the potential role of hBDs in the regulation and maintenance of the skin barrier function. In this review, we describe the current knowledge concerning the role of hBDs in skin barriers and discuss the potential clinical implications of these peptides in cutaneous biology. Understanding the roles of hBDs in the regulation and maintenance of skin barriers may aid in the development of novel therapeutic strategies for skin conditions where the skin barrier is impaired, such as atopic dermatitis and psoriasis.