The cryopreservation of avian semen without significant loss of fertilizing ability is of practical interest to the poultry industry and for the conservation of genetic biodiversity. However, the unique physiological features make avian spermatozoa highly sensitive to damage caused by cryopreservation. The potential proteomic changes in turkey (Meleagris gallopavo) semen throughout the cryopreservation process have never been investigated previously. Therefore, the purpose of the present study was to evaluate the proteomic changes of spermatozoa during cryopreservation at three stages (fresh, equilibrated, and frozen/thawed semen) using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF/TOF). No differentially expressed proteins (DEPs) were found in spermatozoa proteome of fresh and equilibrated semen. One hundred forty six protein spots differed significantly between fresh and frozen/thawed spermatozoa. One hundred three protein spots differed significantly between equilibrated and frozen/thawed spermatozoa. Most of the proteins that changed in response to cryopreservation were related to the generation of precursor metabolites and energy through aerobic respiration, flagellum-dependent cell motility, and binding of sperm to the zona pellucida. This suggests the complexity of the processes leading to decreases in the quality of cryopreserved semen.