To identify tissue-specific expression gene in scrotum of differential scrotal circumference bulls and analyze the function of the specific gene on the development of the bulls scrotum in this study. The DDRT-PCR and Reverse Northern Blot Analysis were used to identify tissue-specific expression gene in 4 bulls with differential scrotal circumference. The results were shown that six genes corresponded to genes of known or inferred function, either the bovine gene or the likely human orthologue and three genes or ESTs were unkown. Those were Bos taurus ebd-P2 pseudogene, Bos taurus similar to F-box only protein 21 isoform2, Bos taurus mRNA for proline-rich protein P-B, Bos taurus similar to Kinesin heavy chain isoform 5C, Bos taurus similar to ankyrin repeat domain protein 15 isoform, and Bos taurus similar to galactosidase, beta 1-like. And Bos taurus ebd-P2 pseudogene and Bos taurus similar to galactosidase, beta 1-like were founded both highly expressed in bulls which had bigger scrotal circumference. Their functions may be involved with sperm maturation in the epididymis, sperm protection and preventing the ascent of microorganisms into the adjacent testes and responsible for converting immature sperm into competent functional cells, and movement of spermatozoa.