The objective of this study was to compare the effects of two different thawing methods (in the vagina of cow or in warm water bath) on post-thaw spermatological parameters in frozen bull semen. A total forty-eight-0.25 mL straws belonging four Simmental bulls were used in this study. While straws were thawed in a water bath at 38⁰C for 25 seconds in warm water thawing (standard) method, they were thawed in the vagina of cow by holding for 3 minutes in vaginal thawing method. Motility and kinematic parameter values of thawed bull semen were evaluated using a computer-assisted sperm analyzer (CASA). In addition, mitochondrial membrane potential of spermatozoa was determined by using flow-cytometry. While the total motility, the rate of sperm with high mitochondrial activity, the rate of rapid spermatozoon and VCL was significantly lower (P<0.05), the rate of dead spermatozoon, LIN and STR was significantly higher (P<0.05) in vaginal thawing method compared to samples thawed in warm water bath. However, there was no statistically significant difference between the two thawing methods in terms of progresif motility, VAP, VSL, WOB, ALH, and BCF (P>0.05). In conclusion, it can provide sufficient spermatological quality after thawing by placing frozen bull semen directly into the insemination catheter and thawing it in the cow's vagina. However, it may useful to determine the effects of individual differences in larger populations and incubation time on post-thaw spermatological characteristics before using this method in field conditions.