Within the human testis, large amounts of sulfated steroid hormones are produced. As shown in breast tissue and placenta, these might not only be excretion intermediates, but re-activated in target cells by steroid sulfatase (STS). This process is called sulfatase pathway and may play a pivotal role in para- and/or intracrine regulation by creating a local supply for steroid hormones. This requires a facilitated transport via uptake carriers and efflux transporters as these hydrophilic molecules cannot pass the cell membrane. Moreover, blood-testis barrier formation in the testis requires a transport through Sertoli cells (SCs) to reach germ cells (GCs). Sertoli cells are therefore expected to play a key role as gate-keepers for sulfatase pathway in human seminiferous epithelium. We analyzed the mRNA and protein expression of uptake carriers and efflux transporters like organic anion-transporting polypeptides (OATP2B1, OATP3A1) and multidrug resistance-related proteins (MRP1, MRP4) in testicular tissue and cultured Sertoli cells (FS1, HSEC). Additionally, expression pattern of STS as well as sulfonating enzymes (SULTs) were assessed. OATP2B1, OATP3A1 and STS were detected in SCs as well as GCs, whereas MRP1 is only expressed in SCs, and SULT1E1 only in Leydig cells, respectively. By transcellular transport of [H]DHEAS in HSEC, we showed a functional transport of sulfated steroids in vitro. Our data indicate that steroid synthesis via sulfatase pathway in Sertoli cells in vivo and in vitro is possible and may contribute to paracrine and intracrine regulation employing the local supply of sulfated and free steroid hormones inside seminiferous tubules.