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This study measured the hypothesis that Rhodiola Sachalinensis Saccharide (RSS) had the positive effect on bovine cryopreservation sperm.RSS was extracted from rhodiola sachalinensis (R.sachalinensis) and added into bovine frozen diluents with 2, 3, 4, 5, 6% (w/v), repectively.After freezing-thawing, the percentages of total motile sperm (TMS), rapid progressive motile spermatozoa(RMPS) of bovine frozen-thawed sperm were significantly higher with the concentrations of 4%(48.24%, 48.43%) and 5% (52.66%, 49.64%) RSS than other groups, respectively (P<0.05).The plasma membrane integrity (PMI) and mitochondrial activity (MA) of bovine frozen-thawed sperm were significantly lower at the concentrations of 2%, 3% and 6% RSS than others (P<0.05),respectively.The values of AI in the groups of 2% and 3% RSS was significantly lower than other groups (P <0.05).Compared with the groups of supplementing 4%, 5% and 6% RSS, the values of MDA were higher in the control group and the concentration of 2%, 3% RSS (P<0.05), and there was no difference among the addition of 4%, 5% and 6% RSS (P>0.05).The contents of vitamin E were significantly lower in control group and groups with the concentrations of 2%, 3%, 4% RSS than the groups with 5% and 6% RSS (P<0.05).The results demonstrated that the comet rate (CR) was improved significantly in the groups with supplemented 4% (29.55%) and 5% (25.80%) RSS (P<0.05).In the groups of adding 2% and 3% RSS, the CR, tail DNA (TD) and tail moment (TM) were increased significantly (P<0.05).