Sumoylation affects multiple cellular events including chromatin inactivation and transcriptional repression. Our data provides the first characterization of SUMO-1 expression during human spermatogenesis using high-resolution cellular SUMO-1-bioimaging. During human meiotic prophase, SUMO-1 localizes to sex chromosomes, and centromeric and pericentromeric chromatin. As human spermatocytes progress toward end of prophase in meiosis I, SUMO-1 is no longer detected within the sex body and peri-centromeric heterochromatin but localizes exclusively to centromeres. SUMO-1 localization along sex chromosome axes, pseudoautosomal region, and centromeres of both chromosomes supports a role for SUMO-1-sumoylation in epigenetic events occurring over the entire sex body, e.g., meiotic sex chromosome inactivation and chromatin condensation. Centromeric SUMO-1 throughout meiotic prophase suggests a role in centromeric chromatin condensation and/or other centromere/kinetochore functions. SUMO-1 is likely involved in both facultative and constitutive heterochromatin processes in spermatocytes. Haploid round spermatids show a consistent association of SUMO-1 with centromeric clusters. During spermatid elongation, SUMO-1 localizes in the manchette perinuclear ring. Steroidogenic Leydig cells show some cytoplasmic but strong nuclear and perinuclear SUMO-1. Peritubular myoepithelial cell SUMO-1 co-localizes with centromeric heterochromatin. In epithelial Sertoli cells, when associated with centromeric heterochromatin, SUMO-1 is adjacent but not co-localized with the nucleolus. Male germ cells demonstrate no SUMO-1-nucleolar association. Human and rodent Sertoli cells consistently show an inverse correlation between androgen receptor and SUMO-1 expression and compartmentalization. Sertoli cells from certain infertile patients, however, showed greatly decreased SUMO-1 and AR. Our data suggest that human testicular SUMO-1 has specific functions in heterochromatin organization, meiotic centromere function, and gene expression.