Abstract: This study was conducted to investigate the effect of semen collection frequency on semen quality of male F3 hybrids of Jersey Cattle and Tibet Yellow Cattle. Twelve 3- to 4-year-old healthy male F3 hybrids of Jersey Cattle and Tibet Yellow Cattle were selected and randomly divided into four groups. In group Ⅰ, the semen samples were collected once every 2 days; in group Ⅱ, the semen samples were collected once daily for two concessive days, and after a 1-day rest, the samples were collected once daily for another two concessive days; in group Ⅲ, the semen samples were collected once daily for three concessive days, and after a 1-day rest, the samples were collected once daily for another three concessive days; in group Ⅳ, the semen samples were collected once, and after a 2-day rest, the samples were collected once again. The semen samples were collected at 9 ： 00 for each group, and the experiment lasted for 2 months. Three duplicates were set in this study. The results showed that there were significant differences in sperm density, sperm motility, sperm acrosomal integrity and sperm GR enzyme activity between group Ⅰ and group Ⅳ （P〈0.05）, and very significant differences in these indicators were also observed between group Ⅰ, group IV and group Ⅱ, group Ⅲ （P〈0.01）. There were significant differences in sperm CAT value between group Ⅰ and group Ⅳ （P〈0.05） as well as between group Ⅱ and group Ⅲ （P〈0.05）, and very significant differences in sperm CAT value were also observed between group Ⅰ, group Ⅳ and group Ⅱ, group Ⅲ （P〈0.01）. The combined data suggest that the best semen quality is obtained in group IV followed by group Ⅰ.