<p>Lysine malonylation (Kmal) is a novel post-translational modification (PTM) implicated in numerous biological processes. Our recent study finds that human sperm proteins are modified by Kmal, but the functional significance of Kmal in human sperm remains unclear. The present study shows that Kmal primarily occurs in human sperm tail proteins with molecular weights ranging from 15 to 250 kDa. Similar to somatic cells, Kmal is derived from malonyl-CoA, with acyltransferase P300 and sirtuin 5 (SIRT5) potentially acting as the writer and eraser, respectively, for Kmal in human sperm. The Kmal level in asthenozoospermic sperm is significantly higher than that in normozoospermic sperm and exhibits a negative correlation with progressive motility. Elevated Kmal levels in asthenozoospermic sperm are associated with reduced sperm SIRT5 and ATP levels, as well as inhibited glycolysis. Furthermore, the induction of sperm Kmal by sodium malonate significantly diminishes the motility and penetration ability of normozoospermic samples by reducing sperm [Ca²⁺]i, ATP, and cAMP levels, and by suppressing glycolysis and PKA activity. Our findings elucidate the regulatory function of Kmal in human sperm motility and its association with asthenozoospermia, thereby offering insights for the diagnosis and treatment of idiopathic male infertility.</p>