Spermatogonial stem cells (SSCs) are vital for lifelong spermatogenesis in man. In their niches, a special growth factor milieu and structural support by surrounding cells are thought to ensure their maintenance. In man, the cells of the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), are considered to contribute to this microenvironment and the overall testicular microenvironment via secreted proteins. Therefore, the secretome of cultured HTPCs from five individual men was analyzed by LC-MS/MS. Quantification and comparison to the proteome of HTPC lysates revealed 263 out of 660 identified secretome proteins to be at least 5-fold enriched in the culture media. To obtain additional evidence for secretion, signal peptide and gene ontology (GO) enrichment analyses were applied. The latter revealed--besides extracellular matrix (ECM) components--a significant over-representation of chemokines and growth factors acting in signaling pathways that appear critical for SSC maintenance. Immunohistochemistry, performed with human testicular sections, depicted expression of selected proteins in vivo. The significant enrichment of proteins related to cell adhesion and migration may indicate their involvement in SSC regulation. Our data strongly support the hypothesis of a crucial role of HTPCs in the composition of SSC niches in man.