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This study aimed to investigate the expression of serine protease inhibitor kazal type 1 (SPINK1) and its carcinogenic effect in oral tongue squamous cell carcinoma (OTSCC). Design: Initially, bioinformatics analysis was conducted using data from The Cancer Genome Atlas and Gene Expression Omnibus to compare SPINK1 mRNA expression between malignant and adjacent tissues. Subsequently, the impact of differential expression on survival and other clinical variables was examined. Additionally, histology microarray analysis was performed to assess SPINK1 protein expression in 35 cases of malignant and adjacent tissues. Finally, alterations in SPINK1 expression were evaluated to determine its biological phenotypes in OTSCC, including proliferation, apoptosis, invasion, and metastasis. Results: OTSCC tissues exhibit higher levels of SPINK1 compared to surrounding cancerous tissues. Notably, increased SPINK1 expression correlates with the pathological N stage and independently predicts overall survival among patients with OTSCC. Conclusion: Suppression of SPINK1 inhibited OTSCC cell proliferation, invasion, and motility while promoting apoptosis. These findings suggest that SPINK1 may serve as a prognostic biomarker as well as a potential therapeutic target for managing OTSCC. • High SPINK1 expression in oral tongue squamous cell carcinoma (OTSCC) is related with the pathological N stage. • SPINK1 serves as a crucial independent factor influencing the overall survival of patients with OTSCC. • SPINK1 promotes proliferation, invasion, and motility of OTSCC cells while suppressing apoptosis. [ABSTRACT FROM AUTHOR]