Previous studies have shown that both density gradient centrifugation (DGC) and swim up (SU) procedures can select spermatozoa with longer telomeres for assisted reproduction techniques (ART). However, it is unknown which approach is more effective. The aim of the present study was to compare the effects of these two methods on sperm telomere length (STL). A total of 150 normozoospermic subjects were recruited. STL, DNA fragmentation index (DFI), reactive oxygen species (ROS) content and progressive motility of semen samples were detected before and after the procedures of DGC and SU. When compared to raw semen, the average length of sperm telomeres was significantly longer after the two sperm preparation methods. However, no significant difference was found between the DGC and SU procedures. We also found that semen prepared by the two methods had lower DNA fragmentation, ROS content and sperm progressive motility. However, no significant difference was found in those parameters between the two procedures. This is the first study that compares the effects of the DGC and SU procedures on STL, and the results show that both methods can recover a sperm population with longer STL and better DNA integrity for ART.