Erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‑TKI), is widely applied as a first‑line treatment for non‑small cell lung cancer (NSCLC) and greatly improves the clinical outcomes of patients. However, acquired resistance to EGFR‑TKIs remains a major clinical challenge. Here, we identified guanylate‑binding protein‑1 (GBP1) as a novel protein related to erlotinib resistance, and explored the specific mechanism by which GBP1 is involved in erlotinib resistance. First, the human NSCLC cells PC9ER and HCC827ER were generated by exposing cells to increasing concentrations of erlotinib over 6 months. We screened different genes between erlotinib‑sensitive and erlotinib‑resistant cells with data from the Gene Expression Omnibus database and detected the expression of these genes in erlotinib‑resistant and erlotinib‑sensitive cells by quantitative real‑time polymerase chain reaction (qPCR). Moreover, we constructed GBP1‑knockdown and GBP1‑overexpressing cells to determine the IC50 value of erlotinib, to perform an apoptosis assay and to examine cell cycle distribution. A subcutaneous tumorigenesis test was used to analyze how GBP1 affects erlotinib resistance. Then, mass spectrometry analysis and coimmunoprecipitation were performed to verify the interaction between GBP1 and phosphoglycerate kinase 1 (PGK1). Changes in epithelial‑mesenchymal transition (EMT)‑related markers were observed following the upregulation and downregulation of PGK1 expression. Finally, a rescue experiment was used to determine whether GBP1 regulates EMT through PGK1. In the present study, GBP1 was significantly upregulated in erlotinib‑resistant cells, compared with erlotinib‑sensitive cells. In vitro and in vivo experiments showed that upregulated GBP1 expression contributed to erlotinib resistance, while decreased GBP1 expression had the opposite effect. As shown by performing survival analysis, high GBP1 expression predicted poor prognosis in patients with lung adenocarcinoma. Furthermore, the interaction between GBP1 and PGK1 was confirmed, and a rescue experiment revealed that GBP1 regulates EMT via PGK1. Finally, functional experiments showed that EMT is involved in erlotinib resistance. Our study suggests that GBP1 regulates erlotinib resistance via PGK1‑mediated EMT signaling, suggesting GBP1 as a potential therapeutic target in erlotinib‑resistant NSCLC.