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The aim of current experiment was to sequence the high-throughput transcriptome of the Xiangxi and Simmental cattle's sperms by using RNA SEQ technology and to screen the expression genes related to the development and reproduction of the sperms. For this purpose, sperms from three Xiangxi yellow cattle and Simmental cattles were collected, separated and purified and cDNA library was constructed. After sequencing, we searched the database, screened the differential expression, analyzed the go and KEGG pathway by David software, and screened the genes related to sperm development through the function inquiry of gene cards. The results of the current study showed that a total of 4890 new genes were discovered, and out of 4890 new gens 2451 were annotated. One hundred fifty-five differentially expressed genes were selected, including 88 up-regulated genes and 67 down-regulated genes. According to the GO analysis, the differentially expressed genes involved in biological processes accounted for 40.9%, cell components accounted for 42.5%, and molecular functions accounted for 16.7%. KEGG pathway analysis showed that the differentially expressed genes were more distributed in the three pathways of signal transduction, cancer-related and neurological diseases. In the up-regulated genes, ten genes that may be directly involved in signal pathway regulation, cell cycle regulation and cell proliferation and differentiation were RICTOR, VDAC2, ETNK1, TRIM36, SET, SRSF5, JMJD1C, ZNF280D, RIOK3 and HDAC1. However, further research is required to explore these findings. [ABSTRACT FROM AUTHOR]