Asthenozoospermia, a prevalent contributor to male infertility, exhibits a multifaceted pathogenesis. This study identified a significant downregulation in sperm dynein heavy chain 3 (DNAH3) protein levels in individuals with asthenozoospermia. To elucidate the role of DNAH3 in asthenozoospermia, we constructed Dnah3-knockout mice, which exhibited asthenozoospermia and sterility. The sperm motility of Dnah3-knockout mice significantly declined compared to wild-type mice. However, spermatozoa from Dnah3-knockout mice displayed normal morphology in hematoxylin and eosin staining and transmission electron microscopy analyses. Sperm metabolomics revealed that DNAH3 deficiency disturbed sperm energy metabolism, resulting in substantial reductions of L-palmitoylcarnitine and glycocholic acid. Notably, offspring were successfully obtained from Dnah3-knockout male mice through intracytoplasmic sperm injection. Collectively, these findings indicate that DNAH3 deficiency induces disturbances in energy metabolism, rather than abnormalities in sperm flagellar morphology, culminating in asthenozoospermia development. Our investigation provides valuable insights into understanding asthenozoospermia and offers guidance for clinical consultation.