Testosterone masculinizes male sexual behavior by providing organizational and activational effects during the perinatal and peripubertal periods and during adulthood, respectively. We revealed that the emission of ultrasonic vocalizations (USVs) and mounting behavior was regulated by different neural circuits. However, the detailed testosterone effects on these two behaviors have not been fully elucidated. Here, we evaluated the time-dependent effects of testosterone on USVs and mounting behavior in mice using a testosterone treatment model, in which females were treated with testosterone to assess the "gain-of-function" and a "loss-of-function" model. In the loss-of-function model, we usedAd4BP/SF-1(Delta FLC/-)male mice, in which testosterone production was abolished in prenatal and postnatal stages, andAd4BP/SF-1(Delta FLC/Delta FLC)mice, in which testosterone production was markedly reduced only in prenatal stages. When testosterone was administered to female mice during the neonatal and peripubertal periods, but not during adulthood, USV emissions increased. Conversely, testosterone treatment in adult female mice increased the mounting behavior, but not USVs. InAd4BP/SF-1(Delta FLC/-)mice, USVs and mounting behavior was completely absent.Ad4BP/SF-1(Delta FLC/Delta FLC)male mice displayed equivalent levels of USVs but less mounting behavior. Collectively, these results suggest that testosterone has dual regulatory roles in USV emissions and mounting behavior.