Testosterone is considered to be released from Leydig cells via passive diffusion because of its hydrophobicity; however, the exact mechanism underlying testosterone secretion and the transporter involved are both unknown. Multidrug and toxic compound extrusion (MATE) transporters are predominantly found in the kidneys and liver and are thought to function in the elimination of metabolic organic cations during the final step of excretion in the kidney. In contrast, mMATE2 has been shown to be predominantly expressed in testicular Leydig cells. Although the physiological function of mMATE2 in Leydig cells is unknown, we hypothesized that mMATE2 acts as a testosterone exporter and is responsible for the secretion of testosterone from Leydig cells. Therefore, in the present study, we investigated the involvement of the MATE transporter in testosterone secretion from pig Leydig cells. Immunohistochemical analysis with anti-pig MATE2 antiserum indicated that the MATE transporter is present in pig Leydig cells. Additionally, treatment with the MATE inhibitors cimetidine and pyrimethamine reduced the testosterone secretion from pig Leydig cells but increased the intracellular testosterone levels. Estradiol release and intracellular estradiol level induced by human chorionic gonadotropin (hCG) further increased with cimetidine treatment. These results indicated that testosterone produced by hCG treatment is secreted from Leydig cells via the MATE transporter; however, in the presence of cimetidine or pyrimethamine, this MATE transporter-mediated secretion was inhibited, resulting in increased intracellular testosterone levels and estradiol production in Leydig cells. Thus, the MATE transporter may be responsible for testosterone secretion from Leydig cells.