Abstract: This study was conducted to investigate the effect of Rhodiola rosea polysaccharide on semen preservation at 4℃ of F3 hybrids of Jersey Cattle and Tibet Yellow Cattle under high-altitude environment. The fresh semen samples were collected from F3 hybrids by artificial vagina method. The basic solution of improved cryoprotective extenders for dairy cow semen was supplemented with 0, 0.02, 0.04, 0.06, 0.08 and 0.10 mg/mL of Rhodiola rosea polysaccharide. After 48 h preservation at 4 ℃, the sperm quality of F3 hybrids semen samples added with different concentrations of Rhodiola rosea polysaccharide were compared.The sperm motility and acrosome integrity rate were used as assessment indexes for semen quality, and the glutathione reductase（GR） activity and catalase（CAT） activity were used as indexes for antioxidative effect evaluation of Rhodiola rosea polysaccharide. The results showed that the 0.08 mg/mL supplementation group has the highest sperm motility（P〈0.01）, and its sperm acrosome integrity rate was significantly higher than that of 0.08 mg/mL and 0.10 mg/mL supplementation groups（P〈0.05）and was extremely and significantly higher than that of the other groups（P〈0.01）; The activity value of GR and CAT in 0.08 mg/mL supplementation group reached 72.39 U/L and 5.79 U/mL respectively and was extremely and significantly higher than that of the other groups（P〈0.01）. The combined data showed that supplementation of 0.08 mg/mL Rhodiola rosea polysaccharide in cryoprotective extenders for dairy cow semen significantly improved the semen quality of F3 hybrids of Jersey Cattle and Tibet Yellow Cattle and enhanced the antioxidative effect of cryoprotective extenders. This research laid a foundation for the production and application of frozen semen for hybrids of Jersey Cattle and Tibet Yellow Cattle, Jersey Cattle and Holstein cows in the future.