Stallion spermatozoa rely on oxidative phosphorylation (OXPHOS) to generate ATP for motility and as such are susceptible to high levels of oxidative damage due to mitochondrial superoxide leakage. As a result, lipid peroxidation is a feature of stallion spermatozoa; however peroxidation was not identified as a major determinant of fertility in prospective trials, suggesting the presence of highly effective defense mechanisms in this species. The primary defense against peroxidation is aldehyde dehydrogenase (ALDH), which is responsible for converting cytotoxic aldehydes such as 4-hydroxynonenal (4HNE), generated as a consequence of lipid peroxidation, to the corresponding carboxylic acid.  The expression of ALDH may be detected with the fluorescent probe Aldeflour^™, a stain originally created for isolating stem cells. The aim of this study was to ascertain whether stallion spermatozoa contain an active form of ALDH, and to characterise the relationship between ALDH expression, lipid peroxidation and motility parameters using Aldeflour ^™, 4HNE antibody staining and CASA respectively. Preliminary proteomic assessment of stallion spermatozoa revealed a number of ALDH isoforms, including ALDH2; a mitochondrial isoform localised in the matrix. Ejaculates from three pony stallions (N=9) were incubated at 37°C for 24 h. Motility (CASA), lipid peroxidation (4HNE), and ALDH activity (Aldefluor™; flow cytometrically as per manufacturer’s instructions) were assessed at 1 h and 24 h. Results revealed an inverse relationship between ALDH activity and 4HNE levels; ALDH positive cells decreased (50.9 to 20.7%), 4HNE positive cells increased (34.2 to 53.4%) and total motility decreased (86.0 to 35.5%). Interestingly, significant correlations between CASA parameters and ALDH positivity were observed (R²=0.62, 0.55, 0.63, 0.5, 0.41, and 0.44 for PM, VSL, % Rapid, VAP, LIN, and VCL respectively), indicating a protective effect of ALDH on sperm functionality. This relationship indicates that the Aldefluor™ probe is a useful tool for the assessment of antioxidant defenses of equine spermatozoa and may provide an upstream predictor of sperm longevity for clinical ART and diagnostic applications.