For commercial aquaculture, the obtainment of all-female salmonid populations is important in fish farms. After freezing process, variable spermatozoa maturity, higher sperm concentration, low sperm quality, and reduced fertilization success have been observed in sex-reversed female rainbow trout. For these reasons, the objective of this study was to assess effect of dilution rate and supplementation of a N-(2-Mercaptopropionyl)-Glycine (MPG)-based extender on sperm motility and fertilizing capacity of sex-reversed rainbow trout (Oncorhynchus mykiss). The supplementation of MPG [0 (control, 0), 1 mM; 2 mM; 4 mM] to extenders and dilution ratio (1:9, 1:15 and 1:25) were tested in sex-reversed female rainbow trout spermatozoa during cryopreservation. For thawing, the straws (0.5 ml) were placed in a water bath at 37°C for 30 s. Our results showed that the best concentration of MPG was 2 mM for post-thaw motility duration (120.67±9.07%), fertilization (62.67±3.10%) and hatching rate (54.33±3.10%) at 1:15 dilution rate. Overall, MPG provided improvements during cryopreservation process and could be used as protective agent.