Grainyhead-like protein 3 homolog (GRHL3) has been identified as a top transcription factor associated with keratinization in lung squamous cell carcinoma (LUSC). We designed this study to elucidate the function of GRHL3 in radioresistance in LUSC and the mechanism involved. Transcriptome differences between radioresistant and parental cells were analyzed to identify the hub transcription factor. GRHL3 expression was overexpressed in radioresistant cells relative to parental cells, and the knockdown of GRHL3 conferred sensitivity to radioresistant LUSC cells, induced DNA damage, inhibited cell survival, and reduced tumor load in mice. GRHL3 promoted ring finger protein 2 (RNF2) transcription by binding to the RNF2 promoter. GRHL3 induced a radioresistant phenotype in parental cells and led to compromised anti-tumor immune responses of CD4+ T cells and NK cells. The GRHL3-promoted tumor progression was reversed by the knockdown of RNF2. The DNA methylation of GRHL3 was reduced in radioresistant cells. All in all, as GRHL3, helps LUSC cells escape from the immune surveillance and mediates radioresistance, it might be an attractive target for therapy-resistant LUSC.Copyright © 2025. Published by Elsevier Inc.