Objective ·To identify the gene expression program (GEP) of cancer-testis genes (CTGs) during spermatogenesis based on single-cell transcriptome data from the testis and investigate their association with the prognosis of cancer patients. Methods ·Expression profiles of normal and tumor tissues were obtained from the GTEx and TCGA databases to screen CTGs. The GEP of CTGs during spermatogenesis was identified by applying the leiden clustering algorithm to testicular single-cell transcriptome data. DecoupleR was used to evaluate the activity levels of GEP and determine the cell types and stages of spermatogenesis where each GEP was active. Subsequently, DecoupleR was used to evaluate the activity levels of GEP in tumor tissues and analyze the correlation between GEP and cancer patient survival. Results ·Based on the expression profiles of normal and tumor tissues from the GTEx and TCGA databases, 917 CTGs were identified. By using the expression patterns of CTGs in the testicular single-cell transcriptome data, seven GEPs were identified through the clustering algorithm. Activity level analysis revealed that GEP5 was active in the early stages of spermatogenesis, including spermatogonia stem cells, differentiating spermatogonia, and early primary spermatocytes. The distribution of GEP5-associated genes was predominantly found on the X chromosome. Additionally, survival analysis demonstrated a statistically significant negative correlation between GEP5 activity levels and patient survival in various tumors. Conclusion ·During spermatogenesis, GEP5 is active in early stages, and its associated genes are primarily located on the X chromosome. In multiple tumor types, the activity level of GEP5 is closely related to patient prognosis.