The application of ultrasonic vibration was performed to modify the water molecules as the main compositions of the freezing medium used for human sperm cryopreservation. Different time periods of ultrasonic vibration (ULV) at the frequency of 28 kHz were applied for the evaluation of physicochemical properties of the water molecules. The most significant bubble size, zeta potential, and pH were obtained for the water molecules exposed to ultrasonic vibrations for 18 minutes and this time period was selected for further experiments due to the optimum results. In the next stage, semen samples were diluted with freezing medium containing ULV-exposed water and then cryopreserved. All the semen parameters were significantly reduced in cryopreserved groups as compared with the fresh control group. The highest percentage of total and progressive motility, viability, membrane and DNA integrity, and mitochondrial membrane potential were observed in frozen ULV compared with the frozen control. The rate of apoptosis in frozen ULV was significantly lower than that of in the frozen control. Furthermore, the gene expression ratios of α- and β-tubulins were significantly increased during cryopreservation, while the expression ratio of the tubulin polymerization promoting protein (TPPP) gene was decreased. Similar results were also observed when the protein levels of the genes mentioned earlier were evaluated by the ELISA method. Therefore, the changes in physicochemical properties of the freezing medium of human sperm cryopreservation using ULV can improve the quality of frozen products.