Objective The aim of this study was to investigate the masculinizing effects of progesterone (PRO) exposure at different concentrations on the morphology of female mosquitofish (Gambusia affinis). Methods Immature female G. affinis individuals were put into static water with 0.5, 5, 50 and 500 nmol/L progesterone (n=56), respectively. In addition, control group and parallel groups were set up. After 42-day-long exposure, we measured four main indexes of the G. affinis: the body length, body weight, morphological changes in the anal fin 3rd fin and the 14, 15 and 16 vertebral ribs. We also observed the state of ovarian development. Results The body lengths (BL) of experimental groups exposed at concentration of 50 and 500 nmol/L progesterone for 42 d showed significant differences, respectively (P<0.01 for both), when compared with those of the control group. The body weights (BW) of experimental groups exposed to progesterone at all concentrations were significantly or very significantly decreased (P<0.05 or P<0.01). Only the progesterone at 5 nmol/L concentration showed significant effect on the body health index (CF) (P<0.05). The section number (FJ) of the anal fin 3rd fin, the section length (FL)and the widest portion width (FW)in the experimental groups had significant difference (P<0.05), respectively, showing significant changes in morphology after the exposure to 500 nmol/L progesterone. However, there was no significant difference between the values resulted by progesterone in other concentrations (P> 0.05). When exposed to 500 nmol/L PRO, the L, D and L:D values of the 14th, 15th and 16th vertebral ribs of the female G. affinis had very significant difference (P<0.01 for all), respectively, indicating significant changes of morphological masculinization. Histological examination revealed that the developmental status of the ovaries of G. affinis in the experimental groups were to different degrees inhibited, and the nuclei appeared swelling in stage Ⅱ and Ⅲ oocytes. Conclusions The results of this study indicate apparent masculinizing effects of progesterone on immature female mosquitofish.