Objective Through the observation of the ultrastructure of testicular sperm and the bioinformatics re-analysis of preliminary laboratory data, to study the mechanism by which the down-regulation of the testicular Clock gene reduces the percentage of forward sperm movement in mouse. Methods Twenty male SPF ICR mice (8 weeks old) were randomly divided into experimental group, the control group, with 6 mice in each group, and the other 2 mice were used as the samples of co-immunoprecipitation experiment. Get  Clock  genes in mice testicular tissue, tem observation of the ultrastructure of sperm. The activity of α-KGDH in the testicular tissue was detected with ELISA, and the transcriptome level of dihydrolipoic acid dehydrogenase (DLD) in the testicular tissue was detected with RT-qPCR. Finally, the interaction between CLOCK protein and DLD protein was verified in testicular tissue of wild-type mice. Results The morphology and arrangement of sperm cell mitochondria in the testis of the experimental group were abnormal. The GO and KEGG enrichment analysis of differential proteins indicated that DLD, one of the subunit constituent proteins of the TCA-cycle's rate-limiting enzyme α-KGDH, was significantly down-regulated in the sperm mitochondria of the experimental group. The α-KGDH enzyme activity in the testis of the experimental group was significantly reduced. The mRNA level of Dld gene in the testis was also significantly down-regulated in the experimental group. And the testis CLOCK protein and DLD protein can interact with each other in the wild type mouse. Conclusion Down regulation of the Clock gene in the testis can lead to changes in the structure and functional protein of mouse sperm mitochondria, which limits the energy output of mitochondria and ultimately leads to a decrease in the percentage of forward sperm movement.