This study aimed to explore the association between the methylation status of the VDAC2 gene promoter region and idiopathic asthenospermia (IAS). Twenty-five IAS patients and 27 fertile normozoospermia (NZ) were involved. GC-2spd cells were treated with different concentrations of 5-aza-2'-deoxycytidine (5-Aza-CdR) for 24 h and 48 h. qRT-PCR was conducted to reveal whether or not VDAC2 expression was regulated by methylated modification. A dual-luciferase activity detection was used to verify VDAC2 promoter activity in GC-2spd cells. Bisulphite genomic sequence was used to analyse DNA methylation of the VDAC2 promoter. The results showed that VDAC2 expression was significantly increased after treated with 5-Aza-CdR. A strong activity of the promoter (-2000 bp to +1000 bp) was detected by dual-luciferase activity detection (P < 0.05). The bisulphite genomic sequencing and correlation analysis showed that sperm motility was positively associated with the methylation pattern of uncomplete methylation and mild hypermethylation, and negatively related to the percentage of moderate methylation. In conclusion, high methylation of the VDAC2 promoter CpGs could be positively correlated with low sperm motility. Abnormal methylation of VDAC2 promoter may be a potential cause to idiopathic asthenospermia.