Abstract: Gobiocypris rarus is listed as a national second-class protected animal and serves as an important aquatic experimental organism. This study aims to establish ultra-low temperature cryopreservation technology for Gobiocypris rarus sperm by investigating the effects of different types and concentrations of extenders, cryoprotectants, and cooling procedures on sperm cryopreservation. Insemination experiments were conducted to assess the quality of cryopreserved sperm. Firstly, the effects of five types of sperm extenders on sperm motility were compared, revealing that sperm in the BSMIS exhibited the highest motility (95.55±2.35)%. The sperm curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and sperm longevity were (103.00±29.82), (84.00±26.21), (126.67±27.57) µm/s, and (37.67±0.58)s, respectively, with no significant difference observed compared to fresh sperm (P>0.05). Secondly, six permeable cryoprotectants at different concentrations were screened, and the highest sperm viability (22.33±2.08)% was achieved when the BSMIS dilution contained 10% methanol. Subsequently, non-permeable cryoprotectants were screened and optimized, with the highest sperm viability (46.00±4.00)% observed when the BSMIS dilution contained 8% methanol and 90 mmol/L glucose. The VCL, VSL, VAP, and longevity were (125.02±7.42), (107.10±4.92), (116.99±6.34) µm/s, and (32.67±2.52)s, respectively, where VSL, VAP were not significantly different (P>0.05) compared to fresh sperm. Finally, the effects of four equilibrium heights and three equilibrium times in liquid nitrogen vapors on the cryopreservation of Gobiocypris rarus were compared. The highest sperm viability was observed when the equilibrium height and time were 3 cm and 10min, respectively. The plasma membrane integrity, mitochondrial activity and DNA integrity of frozen sperm detected by flow cytometry were (55.78±2.13)%, (56.18±0.64)%, and (55.07±2.00)%, respectively. The quality of frozen sperm was further assessed by insemination experiments, and the fertilization and hatching rate of frozen sperm were (87.77±2.18)% and (85.65±4.01)%, respectively, which were not significantly different from that of fresh sperm (P>0.05). These findings showed that the best effect of ultra-low temperature cryopreservation was achieved by placing the sperm of Gobiocypris rarus in liquid nitrogen after equilibration for 10min at an equilibrium height of 3 cm with BSMIS diluent, 8% methanol, and 90 mmol/L dextrose as the cryoprotectant. In this study, the sperm ultra-low temperature cryopreservation technique for the Gobiocypris rarus was preliminarily established to provide technical support for the long-term conservation of its germplasm resources.