<p>Cancer-testis antigens are considered clinically attractive targets for cancer treatment, but their functions and mechanisms are not well elucidated. Here, based on comprehensive bioinformatics analyses, we identify PRAME, a nuclear cancer-testis antigen, as a potential regulator of metastasis in clear cell renal cell carcinoma (ccRCC). Subsequent RNA-Seq and functional studies illustrate that Netrin-4 (NTN4) is a major downstream effector of PRAME, involved in its oncogenic functions. Mechanism analyses reveal that PRAME interacts with the transcription factor CCAAT/enhancer-binding protein beta (C/EBPβ) and the histone methyltransferase enhancer of zeste homolog 2 (EZH2) simultaneously, thereby forming a ternary complex. Subsequently, this complex co-occupies the NTN4 promoter locus, leading to increased trimethylation of histone H3 lysine 27 and epigenetic repression of NTN4 expression, resulting in AKT activation and promotion of ccRCC development. Interestingly, C/EBPβ is characterized to stimulate PRAME expression by binding to the PRAME promoter. Additionally, a cell-permeable peptide has been designed to disrupt the ternary complex and inhibit ccRCC progression in tumor cells and patient-derived xenografts. Thus, our findings not only provide new insights into the prominent role of PRAME in mediating C/EBPβ and EZH2 regulation of NTN4 and tumor metastasis, but also highlight a promising strategy for ccRCC therapy by targeting the C/EBPβ-PRAME-EZH2 complex.</p>