The mule (Equus mulus mulus) is a domestic animal that results from the breeding of a male donkey (Equus asinus) to a female horse (Equus caballus). The number of metacentric and acrocentric autosomal chromosomes in horses and donkeys differs approximately by one-third, resulting in pairing failure of homologous chromosomes during meiosis. Probably for this reason, spermatogenesis in mules rarely advances beyond spermatocytes and leads to sterility in this hybrid equid. The results found in a recent study developed in our laboratory, comparing testis structure and function by using light microscopy in donkeys and mules, suggested that mules’ seminiferous tubules are probably able to sustain complete spermatogenesis. In order to further investigate this hypothesis, we performed an ultrastructural analysis of Sertoli and Leydig cells in both mules and donkeys. The Sertoli cell tight-junction morphology and the morphofunctional integrity of the Sertoli cell barrier were also investigated in these species utilizing lanthanum as a tracer. The results showed that somatic cells of both donkeys and mules had similar structure and organelle morphology. This indicated that Sertoli and Leydig cell structure and function in mules were apparently normal and functional. The same was observed for Sertoli cell tight junctions and the integrity of the Sertoli cell barrier. Taken together, our results support the light microscopy data already obtained indicating that mules are potential viable candidates for transplantation of germ cells originated from donkeys, horses, and other equids.