Variable Charge X/Y (VCX/Y) is a human testis-specific gene family that localized on X and Y chromosomes. In this study, VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST) fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated and the localization of VCY protein in human testis was determined by immunohistochemistry. In the testis seminiferous epithelium, VCY proteins were highly expressed in nuclei of germ cells. Using propidium iodide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainly localized in the nucleoli of COS7 cells. In addition, the colocalization for VCY and VCX-8r in COS7 cells was also observed. With VCY cDNA as bait, a cDNA fragment of acidic ribosomal protein PO was obtained using yeast two-hybrid system. All the information above indicates that VCX/Y protein family might be involved in the regulation of ribosome assembly during spermatogenesis.