Abstract: Objective: To identify key molecular targets in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) and to investigate their potential roles in tumor progression and response to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy. Methods: Differentially expressed genes (DEGs) between EGFR-mutant and EGFR-wild-type lung adenocarcinoma (LUAD) tissues in The Cancer Genome Atlas (TCGA) database were identified using the limma package in R software (P < 0.05, |log2FC| > 1), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. LRRC31 protein expression was examined by Western blotting in different NSCLC cell lines. LRRC31 was knocked down by lentivirus-mediated short hairpin RNA (shRNA), and its effects on the proliferation and migration of EGFR-mutant PC9 and H1975 cells were evaluated using CCK-8, colony formation, and wound-healing assays. Publicly available datasets were further analyzed to assess the association between LRRC31 expression and sensitivity to EGFR-TKIs. The effects of LRRC31 knockdown combined with osimertinib on H1975 cell proliferation were evaluated by CCK-8 and colony formation assays. In addition, LRRC31 was knocked down in osimertinib-resistant H1975/OR cells to assess its association with the response to EGFR-TKI treatment. Results: A total of 73 DEGs were identified, including 42 upregulated and 31 downregulated genes. LRRC31 was significantly upregulated in EGFR-mutant LUAD tissues and in the EGFR-mutant PC9 and H1975 cell lines. In vitro experiments showed that LRRC31 knockdown significantly inhibited cell viability, colony formation, and migration in PC9 and H1975 cells. Database analyses indicated that LRRC31 expression was positively correlated with the IC50 of osimertinib and was upregulated in gefitinib-resistant cells. Functional experiments further showed that, compared with osimertinib treatment alone, LRRC31 knockdown combined with osimertinib exerted a greater inhibitory effect on the proliferation and colony-forming ability of H1975 cells. Moreover, LRRC31 knockdown markedly increased the sensitivity of H1975/OR cells to osimertinib. Conclusion: LRRC31 is highly expressed in EGFR-mutant NSCLC and is associated with malignant tumor phenotypes. LRRC31 knockdown enhances the sensitivity of tumor cells to osimertinib, suggesting that LRRC31 may play a potential regulatory role in the response to EGFR-TKI therapy and in the maintenance of drug-resistant phenotypes. Its potential value as a therapeutic target requires further investigation.