ProAKAP4 is identified within the flagellum of spermatozoa in various mammalian species, serving as a structural protein associated with motility parameters. This investigation focuses on the presence of proAKAP4 in donkey sperm, elucidating its localization, molecular characteristics, and its correlation with motility descriptors and mitochondrial membrane potential. Twelve ejaculates from Catalan donkeys were analyzed in this study. The initial steps involved proAKAP4 sequencing and detection through Western blotting and immunofluorescence. Post-thaw assessments were conducted at 0, 1, and 3 h, encompassing proAKAP4 levels, sperm motility analyzed via Computer-Assisted Sperm Analysis (CASA), and mitochondrial membrane potential determined by flow cytometry using the JC-1 stain. The findings reveal that proAKAP4 in donkeys exhibits a characteristic localization at the principal piece of the flagellum, consistent with observations in other mammals. The molecular weight of proAKAP4 is determined to be 100 kDa. Significantly, a positive correlation (p ≤ 0.05) is established between proAKAP4 concentration and both total and progressive motility. The presence of cryoprotectant is associated with a lower proAKAP4 concentration. Notably, proAKAP4 experiences a substantial decrease (p ≤ 0.05) during the initial hour post-thawing. In conclusion, proAKAP4 is identified in donkey sperm, akin to its presence in other mammals. It exhibits a positive correlation with total and progressive motility, its concentration is notably affected by the presence of cryoprotectant with significant consumption observed during the initial hour following thawing. These findings contribute to our understanding of proAKAP4 dynamics in donkey sperm, providing insights that may have implications for semen preservation and reproductive technologies in equids.