Acipenser schrenckii, commonly known as the Amur sturgeon, is a vital species in China's sturgeon industry due to its significant economic value. Cryopreservation of sperm is a crucial technique for conserving and utilizing sturgeon germplasm resources, facilitating long-term storage of viable sperm. Previous studies have optimized the extender formulas, assessed antioxidant activities, examined enzyme functions related to energy metabolism, and compared sperm ultrastructure. However, there remains a paucity of research on the molecular changes occuring during sturgeon sperm cryopreservation. Addressing this gap, a recent study utilized 4D label-free quantitative proteomics to discern differentially abundant proteins between fresh and freeze-thawing A. schrenckii semen, post-thawing at 24 h. Proteins were extracted using SDT lysis buffer (4% SDS, 100 mmol/L Tris-HCl pH 7.6, 0.1 mol/L DTT) and sonication, followed by centrifugation for collection, identifying a total of 1 859 proteins. Employing a threshold of a 1.5-fold difference factor (FC) and a significance level (P-value) less than 0.05, 200 differentially abundant proteins were delineated, including 92 down-regulated and 108 up-regulated. Subcellular mapping revealed protein localization across various cellular compartments, while GO and KEGG enrichment analyses unveiled involvement in diverse biological processes, cellular components, molecular functions, and signaling pathways. Notably, Fas-activated serine/threonine phosphoprotein kinase 1 (FASTK1) was up-regulated in freeze-thawing semen for 48.81-fold, indicating a role in cell proliferation regulation for self-protection. Conversely, the down-regulation cold inducible RNA-binding protein (CIRP) suggested its involvement in cell proliferation, which ultimately affected the motility of free-thawing semen. Up-regulated Nup proteins potentially contributed to membrane protein conformation changes , destabilizing intracellular environments. Down-regulated DNAH-9 was linked to flagellum membrane disruption and fracture. Changes in proteins associated with energy metabolism and antioxidant response, including CK, SDHC, 26S proteasome, CAT and SOD, may elucidate the decreased motility of sturgeon semen after freeze-thawing process. These molecular alternations may impact sperm viability and function, possibly explain reduced fertilization successpost-cryopreservation. The identified proteins could serve as candidate markers for A. schrenckii semen quality during the freeze-thawing process, guiding the development of improved cryopreservation protocols. This research lays a valuable foundation for deeper exploration into the molecular mechanisms of sturgeon sperm cryopreservation, crucial for the conservation and sustainable utilization of this vital fish resource.