Objective To compare seminal carnitine levels between normal males and asthenozoospermic patients,evaluate its correla-tion with progressive motility(PR)of sperm,and observe the effects of exogenous carnitine supplementation on asthenozoospermic pa-tients.Methods Semen samples were collected from 511 normal fertile males and asthenozoospermic patients.Seminal was measured using a fixed-time assay kit and the levels of carnitine were compared between the two groups.The consistency between seminal carni-tine and PR was assessed.Additionally,77 asthenozoospermic patients received L-carnitine(1 g/time,3 times/day,30 days/course).The levels of seminal carnitine and PR alteration pre-and post-treatment were monitored.Results The seminal L-carnitine level in the patients with asthenospermia([194.34±65.41]μmol/L)was significantly lower than that in normal fertile males([405.43±72.12]μmol/L)(P＜0.01).When the seminal L-carnitine level ≥325 μmol/L was set as the threshold,the statistical results showed that Kappa value was 0.81,with a diagnostic coincidence rate of 93.74％.After one course of administration of L-carnitine,the concentra-tion of seminal L-carnitine([356.03±84.87]μmol/L)and PR([32.69±8.35]％)were significantly higher those that before treat-ment([183.61±79.54]μmol/L and[16.56±7.74]％,P＜0.01).Conclusion The seminal carnitine assay kit could be used for ac-curate and high-throughput quantification of clinical samples,facilitating asthenozoospermia diagnosis and therapeutic efficacy evalua-tion.Exogenous carnitine supplementation may elevate seminal carnitine levels and sperm motility in asthenozoospermic patients and po-tentially improve their fertility.