Abstract Background It has been previously demonstrated that cholesterol content and cholesterol/phospholipid ratio were significantly higher in asthenozoospermia and oligoasthenoteratozoospermia. The majority of published studies have investigated the fatty acid composition of phospholipids rather than lipids themselves. This study evaluated the lipid composition of asthenozoospermic and normozoospermic spermatozoa, and identified the exact lipid species that correlated with sperm motility. Methods A total of 12 infertile asthenozoospermia patients and 12 normozoospermia subjects with normal sperm motility values were tested for semen volume, sperm concentration, count, motility, vitality and morphology. High-coverage targeted lipidomics with 25 individual lipid classes was performed to analyze the sperm lipid components and establish the exact lipid species that correlated with sperm motility. Results A total of 25 individual lipid classes and 479 lipid molecular species were identified and quantified. Asthenozoospermic spermatozoa showed an increase in the level of four lipid classes, including Cho, PE, LPI and GM3. A total of 48 lipid molecular species were significantly altered between normozoospermic and asthenozoospermic spermatozoa. Furthermore, the levels of total GM3 and six GM3 molecular species, which were altered in normozoospermic spermatozoa versus asthenozoospermic spermatozoa, were inversely correlated with sperm progressive and total motility. Conclusions Several unique lipid classes and lipid molecular species were significantly altered between asthenozoospermic and normozoospermic spermatozoa, revealing new possibilities for further mechanistic pursuits and highlighting the development needs of culture medium formulations to improve sperm motility.