In the current study, a total of six 5-year-old healthy bulls reared in heat stress zone were used and semen from these bulls was collected by artificial vagina and undergoes for semen quality analysis. Results of semen quality parameters showed that the average semen volume and sperm density of Simmental bulls were 9.3mL and 1.23 × 10⁹ mL, and sperm acrosome integrity rate were lower as compared to Xiangxi cattle bulls. The transcriptome analysis revealed a 36.81Gb clean data and the obtained clean data of each sample was reached 5.84Gb. The transcriptome analysis further revealed that the percentage of q30 base was 91.19% or above. Based on the comparison results, 4,890 new genes were found, including 2,451 functional annotations. Go analysis explored that GO annotation system included 3 main sections that were biological processes, molecular functions and cellular components, respectively. In conclusion, results showed that the average semen volume and sperm density of Simmental bulls was higher and sperm motility and sperm acrosome integrity rate was lower as compared to Xiangxi cattle bulls. This study has developed a transcriptomic profile of spermatozoa of Xiangxi cattle and Simmental cattle bulls and reported Xiangxi cattle and Simmental cattle bull’s spermatozoa change the gene expression involved in various biological process. This study could provide the experimental basis of poor fertility in Xiangxi cattle bulls and provide possible fertility biomarkers.