This study investigates the effect of de-SUMOylated hexokinase 1 (HK1) degradation on sperm capacitation in vitro. Sperm from adult Landrace pigs was divided into three groups: non-capacitated group, capacitated group, and 16 mM 2-deoxy-D-glucose (2-DG) group. Immunoprecipitation was performed to detect the binding levels of HK1 and small ubiquitin-like modifier 1 (SUMO1) before and after capacitation. Western blotting was used to assess the degradation pathways of HK1 and tyrosine phosphorylation levels after capacitation. Immunofluorescence was employed to localize HK1 and SUMO1 in sperm. A kit was used to measure pyruvate and adenosine triphosphate (ATP) content. Fluorescent staining was used to detect reactive oxygen species (ROS) levels, zinc (Zn²⁺) levels, calcium (Ca²⁺) levels, and mitochondrial membrane potential (MMP) in sperm. Hoechst 33342 staining was used to visualize sperm nuclei and assess sperm-oocyte adhesion rates. The results showed that inhibition of HK1 activity significantly reduced pyruvate and ATP content, sperm capacitation markers (tyrosine phosphorylation, Zn²⁺), MMP, and sperm adhesion rates (P <
0.05), while significantly increasing ROS levels in capacitated sperm (P <
0.01). Immunoprecipitation and Western blotting results indicated that HK1 undergoes de-SUMOylation during capacitation and is selectively degraded via the ubiquitin-proteasome pathway (P <
0.01). Our findings suggest that de-SUMOylated HK1 activates the glycolytic pathway through ubiquitin-proteasome pathway (UPP) degradation, thereby promoting sperm capacitation. These results provide new theoretical insights into the molecular mechanisms of sperm capacitation.