Abstract: Silurus lanzhouensis Chen is an endangered indigenous fish in the middle and upper reaches of the Yellow River. In order to protect its germplasm resources and promote the breeding of new varieties, the cryopreservation procedures and different antifreeze of semen in S. lanzhouensis were studied. The effects of cryopreservation and the damage of cryopreservation were evaluated by thawing activation rate, nuclear DNA detection, scanning electron microscopy and transmission electron microscopy. The results showed that the best sperm cryopreservation procedure were 10% Dimethyl sulfoxide (DMSO) as antifreeze, equilibrated at 4℃ for 20min and –80℃ for 30min, then immediately placed in liquid nitrogen for ultra-low temperature preservation, and thawed sperm in 40℃ water bath, ultimately the sperm activation rate reached (75.56±3.91)%. The results of DNA damage detected by SCGE showed that there was no significant difference in comet rate and damage coefficient of cryopreserved semen of S. lanzhouensis for 10, 20 and 30 days. Scanning electron microscopy showed that the sperm of S. lanzhouensis belonged to a single flagellum type, without acrosomes and lateral fins, and was clearly divided into three parts: the head, the middle and the tail. Centrioles were perpendicular to each other in a “T”-shape, and flagella were typical “9+2” microtubule structures. Structural damage was mainly manifested as membrane damage, cytoplasm and chromatin membrane damage, wrinkle, vesicularization or overall shedding, the gap between the nucleus and the plasma membrane was enlarged, the nucleus was deformed and dispersed, the mitochondrial structure was diffused, and the mitochondrial contents flowed out. The neutrophil complex was translocated, the adventitia of the flagella was deformed and detached, the mid-position was broken, and the microtubule structure was basically intact. The selected cryopreservation technology can effectively preserve the semen of S. lanzhouensis for a long time, and provide theoretical and technical basis for protecting germplasm resources of S. lanzhouensis and improving cryopreservation technology of semen in the future.