3'-Azido-3'-deoxythymidine (AZT), an antiretroviral drug, is often adopted in the therapy for human immunodeficiency virus (HIV) infection, and the characteristics of AZT transport at the blood-testis barrier (BTB) were investigated in this study. In the integration plot analysis that evaluates the transport activity in vivo, the apparent influx clearance of [3H]AZT was significantly greater than that of [14C]D-mannitol, a non-permeable paracellular transport marker. In the uptake study in vitro with TM4 cells derived from mouse Sertoli cells, [3H]AZT uptake exhibited a time- and concentration-dependent manner, of which Km and Vmax values being 20.3 µM and 102 pmol/(min·mg protein), respectively. In the inhibition analysis, [3H]AZT uptake was not affected by extracellular inorganics and some substrates of transporters putatively involved in AZT transport. In the further inhibition analyses to elucidate the characteristics of AZT transport, [3H]AZT uptake was strongly reduced in the presence of several nucleosides, that are categorized as 2'-deoxynucleosides with pyrimidine, whereas little effect on [3H]AZT uptake was exhibited in the presence of other nucleosides, nucleobases, and antiretrovirals. These results suggest the influx transport of AZT from the circulating blood to the testis, and the involvement of carrier-mediated process at the BTB, which selectively recognizes 2'-deoxynucleosides with a pyrimidine base.