The rise of male fertility disorders these last few years appears to be linked to the increase of pollutants and other endocrine disruptors in our environment. Some of these factors can mimic or alter estrogen signaling. Therefore it is essential to better understand the role of estrogens in the male gamete production, in the aim to preserve fertility. Estrogens are steroid hormones produced by the aromatization of androgens through the enzyme aromatase, encoded by the CYP19A1 gene. Although they are usually referred as “female sex hormones”, estrogens are also produced in the adult testis. In mammalian species where CYP19A1/Aromatase expression has been studied, it is noteworthy that the localization of estrogen production varies. The rabbit is an important biomedical model and, to date, the role of estrogens in rabbit testis function is unknown. First, we identified cells expressing CYP19A1 and we showed that testicular estrogens are produced within the seminiferous tubules by germ cells. Then, testicular estrogen and androgen levels were quantified by gas chromatography/mass spectrometry (GC/MS), comparing wild-type and CYP19A1-/- rabbits generated by CRISPR/Cas9 technology. We showed that CYP19A1-/- testes were devoid of 17b-estradiol, while testosterone or androstenedione levels were not modified. As fertility was decreased in male CYP19A1-/- rabbits lacking estrogens (reduction of the number of pups per litter), we investigated several aspects, such as testis histology, DNA methylation of germ /sperm cells and sperm parameters. In particular, CYP19A1-/- males showed reduced sperm motility associated with increased flagellar defects. To highlight estrogen effects during spermatogenesis, high throughput transcriptomic studies are in progress.