Abstract In sites of inflammation or tissue injury, hyaluronic acid (HA), ubiquitous in the extracellular matrix, is broken down into low m.w. HA (LMW-HA) fragments that have been reported to activate immunocompetent cells. We found that LMW-HA induces activation of keratinocytes, which respond by producing β-defensin 2. This production is mediated by TLR2 and TLR4 activation and involves a c-Fos-mediated, protein kinase C-dependent signaling pathway. LMW-HA-induced activation of keratinocytes seems not to be accompanied by an inflammatory response, because no production of IL-8, TNF-α, IL-1β, or IL-6 was observed. Ex vivo and in vivo treatments of murine skin with LMW-HA showed a release of mouse β-defensin 2 in all layers of the epidermal compartment. Therefore, the breakdown of extracellular matrix components, for example after injury, stimulates keratinocytes to release β-defensin 2, which protects cutaneous tissue at a time when it is particularly vulnerable to infection. In addition, our observation might be important to open new perspectives in the development of possible topical products containing LMW-HA to improve the release of β-defensins by keratinocytes, thus ameliorating the self-defense of the skin for the protection of cutaneous tissue from infection by microorganisms.