Exposure to bisphenol A (BPA) has been shown to impact human sperm quality. The epigenetic mechanisms underlying the effect remain unknown. The acetylcholinesterase (ACHE) gene is a sperm-expressed gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase and participates in the apoptosis of cells, including sperm. This study aimed to examine whether BPA exposure is associated with the hydroxymethylation level of the sperm ACHE gene. A total of 157 male factory workers were studied, among whom 74 had BPA exposure in the workplace (BPA exposure group) and 83 had no BPA exposure in the workplace (control group). Urine samples were collected for BPA measurement and semen samples were collected to assay for ACHE hydroxymethylation. Sperm ACHE hydroxymethylation level was higher in the BPA exposure group (p = 0.041) compared to the control group. When subjects were categorized according to tertiles of detected BPA level, higher ACHE hydroxymethylation levels were observed for the lowest, middle, and top tertiles compared to those with BPA below the limit of detection (LOD). In a linear regression analysis adjusted for confounders, a positive linear association between urine BPA concentration and 5-hydroxymethylcytosine (5hmC) rate of the sperm ACHE gene was observed, although the association did not reach statistical significance in all categories after being stratified by the BPA tertile. In conclusion, 5hmC of the sperm ACHE gene was positively associated with BPA exposure, which may provide supportive evidence for BPA’s effects on male fertility or other health endpoints.