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Simple Summary The survival rate of oral squamous cell carcinoma (OSCC) patients has not been improved in the past few decades, likely as a result of a lack of therapeutic targets. Through next generation sequencing for tumor tissues of OSCC patients, the gene expression level of guanylate binding protein 5(GBP5) was significantly elevated in tumor tissues compared with adjacent normal tissues and associated with poor prognosis in OSCC patients. Moreover, we found that GBP5 modulated cell cycle, invasion/migration, and cancer stemness in OSCC cells. Our study indicated that GBP5 might be a potential biomarker and therapeutic target for OSCC patients. Abstract Guanylate binding protein 5 (GBP5) is the interferon (IFN)-inducible subfamily of guanosine triphosphatases (GTPases) and is involved in pathogen defense. However, the role played by GBP5 in cancer development, especially in oral squamous cell carcinoma (OSCC), is still unknown. Herein, next-generation sequencing analysis showed that the gene expression levels of GBP5 were significantly higher in OSCC tissues compared with those found in corresponding tumor adjacent normal tissues (CTAN) from two pairs of OSCC patients. Higher gene expression levels of GBP5 were also found in tumor tissues of 23 buccal mucosal squamous cell carcinoma (BMSCC)/14 tongue squamous cell carcinoma (TSCC) patients and 30 oral cancer patients from The Cancer Genome Atlas (TCGA) database compared with those in CTAN tissues. Immunohistochemical results showed that protein expression levels of GBP5 were also higher in the tumor tissues of 353 OSCC patients including 117 BMSCC, 187 TSCC, and 49 lip squamous cell carcinoma patients. Moreover, TCGA database analysis indicated that high gene expression levels of GBP5 were associated with poor overall survival in oral cancer patients with moderate/poor cell differentiation, and associated with poor disease-free survival in oral cancer patients with moderate/poor cell differentiation and lymph node metastasis. Furthermore, GBP5-knockdowned cells exhibited decreased cell growth, arrest at G1 phase, and decreased invasion/migration. The gene expression of markers for epithelial−mesenchymal transition and cancer stemness was also reduced in GBP5-silenced oral cancer cells. Taken together, GBP5 might be a potential biomarker and therapeutic target for OSCC patients, especially for those with poor cell differentiation and lymph node metastasis.