OBJECTIVE: To analyze the expression levels, clinical significance, Immune infiltration and prognostic value of PRSS53 (Protease Serine 53) in clear cell renal cell carcinoma (ccRCC) using bioinformatics methods. METHODS: Data on PRSS53 in ccRCC were extracted from databases and platforms, including The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression Project (GTEx), The Gene Expression Omnibus (GEO), Xiantao Academic Tool, Human Protein Atlas (HPA) and so on. We analyzed the relationship between PRSS53 expression and the clinical and pathological characteristics, diagnosis, immune infiltration and prognosis in ccRCC patients. Additionally, immunohistochemical analysis was performed on 9 pairs of ccRCC patient samples. RESULTS: PRSS53 was significantly upregulated in ccRCC and was closely associated with the TNM stage and histological grade of ccRCC. Receiver operating characteristic (ROC) curve analysis demonstrated the excellent diagnostic performance of PRSS53 in ccRCC (AUC = 0.928). Patients with high PRSS53 expression exhibited lower overall survival (OS) and disease-specific survival (DSS). Gene set enrichment analysis (GSEA) revealed that PRSS53 is involved in cellular functions such as anchored component of membrane, basement membrane and RNA-binding involved in post-transcriptional gene silencing. Single-sample GSEA (ssGSEA) indicated a positive correlation between PRSS53 expression and T helper cells infiltration levels, and a negative correlation with T gamma delta (Tgd) cell infiltration. PRSS53 was predominantly expressed in renal proximal tubules. The immunohistochemical results and HPA database showed that PRSS53 protein expression was significantly lower in clinical ccRCC tissues compared to normal tissues. CONCLUSION: PRSS53 is a new prognostic biomarker and potential therapeutic target for ccRCC.