In healthy men, several layers of inconspicuously flat cells and extracellular matrix (ECM) proteins build the wall of the seminiferous tubules. The cells of this wall, peritubular cells, are not well characterized. They are smooth-muscle-like and contractile and transport immotile sperm, a function important for male fertility. However, their full functional importance, especially their potential contribution to the paracrine regulation of the male gonad, is unknown. In men with impaired spermatogenesis, the architecture of the tubular wall is frequently altered. Deposits of ECM and morphological changes of peritubular cells imply that functions of peritubular cells may be fundamentally altered. To be able to study human peritubular cells and their functions, a culture method was established. It is based on small biopsies of patients with obstructive azoospermia but normal spermatogenesis (human testicular peritubular cells, HTPCs) and non-obstructive azoospermia, impaired spermatogenesis, and testicular fibrosis (HTPCFs). Results obtained from cellular studies and parallel examinations of biopsies provide insights into the repertoire of the secretion products, contractile properties, and plasticity of human peritubular cells. They produce ECM components, including the proteoglycan decorin, which may influence paracrine signaling between testicular cells. They may contribute to the spermatogonial stem cell niche via secreted factors. They are regulated by mast cell and macrophage products, and in response produce factors that can fuel inflammatory changes. They possess a high degree of plasticity, which results in hypertrophy and loss of contractile abilities. The data collectively indicate important roles of inconspicuous testicular peritubular cells in human male fertility and infertility.