Asthenozoospermia is a common cause of male infertility, the aetiology of which remains unclear in 50–60% of cases. The current study aimed to characterize metabolic alterations in asthenozoospermic seminal plasma and to explore the signalling pathways involved in sperm motility regulation. At first, high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry was used to detect the targeted metabolic network of arachidonic acid (AA). Metabolomic multivariate data analysis showed significant distinction of AA metabolites between asthenozoospermic and healthy seminal plasma. AA as well as its lipoxygenase (LOX) and cytochrome P450 metabolites were found to be abnormally increased, while cyclooxygenase (COX) metabolites were complicatedly disturbed in asthenozoospermic volunteers compared with those in healthy ones. In vitro experiments and western blot analysis of sperm cells revealed a decrease in sperm motility and upregulation of sperm phosphor-P38 induced by AA. P38 inhibitor could increase AA-reduced sperm motility. Also, all the inhibitors of the three metabolic pathways of AA could block AA-induced P38 mitogen-activated protein kinase (MAPK) activation and further improve sperm motility. We report here for the first time that an abnormal AA metabolic network could reduce sperm motility via P38 MAPK activation through the LOX, cytochrome P450 and COX metabolic pathways, which might be an underlying pathomechanism of asthenozoospermia.