OBJECTIVE:Laryngopharyngeal reflux (LPR) is a common affliction that contributes to laryngeal inflammation, symptoms that impact quality of life, and life-threatening illnesses such as cancer. Effective treatment strategies for LPR are lacking. Pepsin is a proinflammatory and carcinogenic element of refluxate. Investigation of molecular pathways involved in pepsin-mediated damage may lead to identification of novel biomarkers and therapeutic targets for LPR. In this study, RNA sequencing was used to examine changes in human laryngeal epithelial cells following brief pepsin insult. Cells were immortalized to generate a model to aid future study of laryngeal injury and therapeutics. STUDY DESIGN:In vitro translational. METHODS:Laryngeal epithelial cells were cultured from a patient without signs or symptoms of LPR or laryngeal cancer. Cells were treated with 0.1 mg/ml pepsin for 1 hour or normal growth media (control) prior to RNA sequencing. Cells were immortalized via HPV E6/7 and characterized by microscopy, immunohistochemistry, G-banding, and soft agar assay. RESULTS:Three hundred ninety-seven genes exhibited differences in expression with pepsin treatment (P < .05). Pathway analysis revealed association with cancer and related signaling processes including dysregulation of cancer-associated molecules, Metastasis-Associated Lung Adenocarcinoma Transcript 1 and KRT82, and the long-noncoding RNA, lipoprotein receptor-related protein 1 (LRP1)-AS, which regulates the putative pepsin receptor LRP1. CONCLUSIONS:A single, brief exposure to pepsin activated cancer-associated signaling pathways in laryngeal cells in vitro, revealing novel mechanisms by which chronic reflux may contribute to carcinogenesis. The cell line developed herein represents a novel tool in which to investigate pepsin-dysregulated pathways identified by RNA sequencing and disparities of tumor proneness of laryngeal subsites. LEVEL OF EVIDENCE:N/A Laryngoscope, 131:121-129, 2021.