Purpose <p>To create a novel medium that retained human sperm quality following cryopreservation at a higher level than that seen with currently available commercial cryoprotectants.</p> Methods <p>Cryopreservation was achieved via 1:1 dilution with cryoprotectant followed by slow-programmed freezing. A NaCl-free cryopreservation carrier medium based on the use of histidine as the major osmolyte was designed that was capable of sustaining human sperm motility over 6&#xa0;days at ambient temperature. This medium was supplemented with ethylene glycol, glycerol, and DMSO to create the basis for a novel cryopreservation medium. Dose-dependent studies with various supplements were then conducted to optimize the effectiveness of this formulation including assessments of vitamin C, EDTA, crocin, zinc, ergothioneine, and <i>myo</i>-inositol, as well as the potential replacement of DMSO by Cyrene™. Post-thaw samples were assessed for motility, vitality, and DNA integrity and then reassessed following sperm isolation with the Felix™ System.</p> Results <p>The completed cryopreservation formulation comprised 4.5% ethylene glycol, 4.5% glycerol, 1% DMSO in a carrier medium supplemented with 0.4&#xa0;mM vitamin C, 1&#xa0;mM EDTA, and 22&#xa0;mM <i>myo</i>-inositol. Spermatozoa frozen in this medium and isolated using the Felix™ System had significantly greater total motility, progressive motility, vitality, and DNA integrity than spermatozoa frozen in a commercially available product that is widely used in infertility clinics.</p> Conclusion <p>A novel cryopreservation medium has been developed in this study that represents a significant improvement over existing technologies.</p>