Fatty acid based lipidomic analysis has been widely used to evaluate health status in human medicine as well as in the veterinary field. In equine species there has been a developing interest in fertility and sperm quality of the stallions. Fatty acids being the principal components of the membranes play an active role in the regulation of the metabolic activities and their role on the spermiogenesis seem to be of great importance for the resulting quality of the sperm and as a consequence of the fertility of the animal. With the application of widely used lipidomic techniques, this study was aimed to: a) evaluate for the first time the fatty acid content of spermatozoa’s membranes of 26 healthy male Martina Franca donkeys and its possible influence on the sperm parameters and b) the evaluation of the composition of the red blood cells’ membrane. For both the spermatozoa and the erythrocytes, the PUFA omega-6 are the principal components of the Total PUFA content (40.38% and 47.79% respectively), however on the spermatozoa’s membranes DPA is the predominant one (27.57%, ) while in the erythrocyte’s membranes DPA is not present and linoleic is the one found in higher concentrations (46.28%). For the spermatozoa, stearic acid levels as well as EPA have demonstrated a positive correlation with motility (%). Progressive motility (%) is positively associated with oleic acid. the data obtained offer information on the structure of the sperm cells as well as the erythrocytes of healthy male MF donkeys and are reflecting the metabolic transformations of the spermatozoa’s membrane during the maturation period. These results provide a deeper understanding of the distribution of the fatty acid content on the spermatozoa’s membrane and their role in sperm parameters that are highly associated with fertility.