Although stallion spermatozoa produce significant quantities of reactive oxygen species, a lag between 4-hydroxynonenal (4HNE) adduction and the loss of motility in stallion spermatozoa suggests the presence of a robust aldehyde detoxification mechanism. Because there is a paucity of studies characterizing the role of aldehyde dehydrogenase (ALDH) in sperm functionality, the aim of this study was to ascertain the relationship between 4HNE production and motility and ALDH expression by stallion spermatozoa. PCR analysis revealed the presence of the ALDH1A3, ALDH1B1, and ALDH2 isoforms in these cells. Strong correlations (P < 0.001) were found between ALDH expression and various motility parameters of stallion spermatozoa including the percentage of progressive (r = 0.79) and rapidly motile (r = 0.79) spermatozoa, whereas repeated measurements over 24 h revealed highly significant correlations among progressive motility loss, 4HNE accumulation, and ALDH expression (P ≤ 0.001). ALDH inhibition resulted in a spontaneous increase in 4HNE levels in viable cells (21.1 ± 5.8% vs. 42.6 ± 5.2%; P ≤ 0.05) and a corresponding decrease in total motility (41.7 ± 6.2% vs. 6.4 ± 2.6%; P ≤ 0.001) and progressive motility (17.0 ± 4.1% vs. 0.7 ± 0.4%; P ≤ 0.001) of stallion spermatozoa over 24 h. Similarly, inhibition of ALDH in 4HNE-challenged spermatozoa significantly reduced total motility over 4 h (35.4 ± 9.7% vs. 15.3 ± 5.1%, respectively; P ≤ 0.05). This study contributes valuable information about the role of the ALDH enzymes in the maintenance of stallion sperm functionality, with potential diagnostic and in vitro applications for assisted reproductive technologies.