Danazol, a derivative of testosterone, is useful for treatment of endometriosis as well as pretreatment for in vitro fertilization and embryo transfer, although its mechanisms of action are unclear. The aim of this study was to investigate the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2k) underwent transplantation of C57BL/6 male (H2b) hearts and received a single dose of danazol (0.4, 1.2 or 4 mg/kg/day) by intraperitoneal injection on the day of transplantation and for 6 days thereafter. An adoptive transfer study was performed to determine whether regulatory cells were generated. The median survival time (MST) of allografts in danazol-treated (1.2 and 4 mg/kg/day) mice was 28 and 63 days, respectively, compared with 7 days in untreated mice. Moreover, secondary CBA recipients given whole splenocytes or CD4+ cells from primary danazol-treated (4 mg/kg/day) CBA recipients 30 days after transplantation had prolonged allograft survival (MSTs, 29 and 60 days, respectively). Cell proliferation, interleukin (IL)-2 and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed allo-proliferation in a mixed leukocyte culture. Flow cytometry showed an increased CD4+ CD25+ Foxp3+ cell population in splenocytes from danazol-treated mice. Danazol prolongs cardiac allograft survival and generates regulatory CD4+ cells.