Abstract Genotoxic therapies such as ionizing radiation eliminate cancer cells by inducing extensive DNA damage but often cause normal tissue toxicity, including cutaneous injury. Extrachromosomal circular DNA (eccDNA) refers to circular DNA fragments outside the chromosomal context, with their formation and persistence linked to DNA damage repair and genomic instability. Despite growing recognition of eccDNA in oncogenesis, its role under genotoxic stress in normal tissues remains poorly understood. Here, eccDNA is profiled in irradiated rat skin using Circle‐seq, identifying alterations in eccDNA number and composition. Specifically, radiation induced circle 17:44148731‐48208624 , in which vacuolar protein sorting 41 homolog (VPS41) is the sole radiation‐induced amplification gene by semiquantitative PCR and gel electrophoresis. The findings show that eccDNA or VPS41 overexpression reduces radiation‐induced skin injury (RISI) in vitro and in vivo. Proteomic and interaction analyses identified metastasis suppressor kangai‐1 (KAI1) as a VPS41‐interacting partner. Notably, VPS41 overexpression promotes KAI1 lysosomal degradation, protecting against radiation‐induced apoptotic cell death. Peptide array analysis pinpoints the VPS41‐KAI1 interaction through the K263 residue, consistent with AlphaFold prediction. The findings uncover a novel mechanism in which radiation‐induced eccDNA, specifically VPS41, mitigates skin injury by modulating KAI1 degradation. This study highlights the role of eccDNA in cellular defense, providing strategies to enhance tissue resilience to genotoxic stress.