The cathelicidin antimicrobial peptide LL37 is a critical element in host defense but inappropriate expression breaks tolerance to self-nucleic acids and promotes inflammatory skin diseases such as psoriasis and rosacea. To understand the mechanism responsible for LL37-dependent inflammation we performed RNA-Seq on human keratinocytes (NHEK) exposed to LL37 and synthetic U1 RNA (U1), a self non-coding RNA released upon tissue damage. Compared to LL37 or U1 RNA alone, the transcriptional response of NHEK to the combination of both LL37 and U1 was unique and included a notable Type 1 interferon signature (167 genes were uniquely increased by 2-fold or more). Screening of a peptide library derived from LL37 showed that the ability of LL37 to penetrate membranes was not necessary for breaking immune tolerance. Proximity ligation assay (PLA) revealed that LL37 facilitated binding of U1 to scavenger receptors on NHEK and macrophages. Use of siRNA against scavenger receptors disrupted this binding and inhibited the inflammatory cytokine response to the combination of LL37 and U1 (P < 0.01 in qPCR, P < 0.001 in PLA). This suggested a 3-way binding interaction with scavenger receptors, LL37 and U1 was required. Inhibitors of endocytosis further established that the ability of LL37 to stimulate expression of IL-6 and interferon-β1 was dependent on clathrin-mediated endocytosis. With this information, a library screen showed binding could be blocked by a competitive peptide and resulted in inhibition of the cytokine and interferon response to LL37 and U1. Analysis of psoriatic lesional skin showed that the binding of LL37 to scavenger receptors occurs in human skin. These results demonstrate that the inflammatory activity of LL37 is mediated by a cell surface, receptor-dependent interaction that is potentially therapeutically targetable.