Infertility has become one of the most common chronic diseases among reproductive- aged individuals, and male factors account for about 50 %. Zinc is a trace element that is essential for sperm quality and male reproductive system. Although several current studies with small sample sizes have investigated this relationship, the conclusions have been still controversial. Here, using a large population sample involved 25,915 participants from Changzhou Maternity and Child Health Care Hospital, we revealed an inverted “U”-shaped trend between seminal plasma zinc concentrations and sperm motility PR/PR + NP (PR, progressive motility; NP, non-progressive motility). The results showed that the highest values of sperm PR/PR + NP observed in the group with intermediate concentrations of zinc (Group 2, 0.25–2.11 mmol/L). The mean values were 43.17 ± 19.03 % and 56.64 ± 20.28 %, respectively. And the lowest values came out in the highest zinc levels group (Group 4, > 3.04 mmol/L). In vitro cell experiments also showed that zinc caused dose-dependent cytotoxicity for GC-2 cells at a threshold value. Furthermore, RNA-seq analysis demonstrated that high concentrations of zinc exerted toxic effects on GC-2 cells through immune injury. Taken together, our findings suggested that moderate amounts of zinc are crucial for human reproduction and excessive concentrations may have adverse effects on male fertility.