Mobile phones play an irreplaceable role in modern people's lives. However, the radiofrequency electromagnetic radiation produced by mobile phones has also caused increasing concern. A cross-sectional study was conducted to investigate the effect of radiofrequency electromagnetic radiation produced by mobile phones on semen parameters in 1634 men who underwent semen examination at the Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, China. Analysis of variance and multivariate linear regression were used to explore differences among different groups. A P <0.05 was considered statistically significant. The results showed significant associations among different groups of daily mobile phone use time and daily duration of phone calls in the percentage of progressively motile spermatozoa (P =0.004 and P =0.007), rapid progressively motile spermatozoa (P =0.012 and P =0.006) and total motile spermatozoa (P =0.004 and P =0.046). After adjustments for the confounding effects of age and body mass index by multiple linear regression, the results showed that the daily duration of mobile phone use had a negative effect on sperm motility. However, there was no statistically significant correlation between daily phone call duration and sperm motility. Therefore, the daily duration of mobile phone use may negatively affect sperm motility and impair male fertility.