Objective: The aim of this work was to evaluate the enzymatic activity of lactate dehydrogenase C4(LDH-C4) as well as the expression level in idiopathic infertile men. Materials and methods: Semen sample from 416 idiopathic infertile men(aged 22 to 45) were collected and processed according to WHO Laboratory Manual for the Examination and Processing of Human Semen(Fifth Edition), and sperms from 30 fertile men was used as normal controls. The evaluation of LDH-C4 enzymatic activity of the spermatozoa was enabled by utilizing a specific LDH-C4 staining method constructed previously, in which the staining working solution contains 2-Hydroxypenternic acid used as LDH-C4 specificity sustrate. Expression of LDH-C4 protein in low activity patients was assessed by western blotting. Immunofluorescence and immunocytochemistry assays were performed in analyzing the sub-cellular location of LDH-C4 in spermatozoon as well as its expression level. Results: Among the 416 idiopathic infertile men, 42 patients(42 of 416) were found with low LDH-C4 enzymatic activity, of which, the evaluation scores were under the normal reference range. Moreover, 38 of the 42 patients revealed a parameter PR less than 30%. In addition, immunofluorescence and immunocytochemistry assays displayed that LDH-C4 located mainly in the neck of the spermatozoon, with a lower expression level in low activity group. Western blotting revealed that expression of LDH-C4 in low activity group(n=36) was significantly lower than that of normal activity controls(n=27), with P<0.05. Conclusions: Abnormal LDH-C4 activity account for more than 10% of all idiopathic infertile cases, and decreased LDH-C4 expression contributed to its abnormal enzymatic activity in idiopathic infertile males.