DMRT1 is a transcription factor containing a DNA-binding domain (DM domain) highly conserved across evolution. It shows gonad-specific expression and it is considered as the Testis Determining Factor in many non-mammalian species. In mice, Dmrt1 disruptions had no impact on testicular differentiation during fetal development, whereas in humans, mutations induce gonadal dysgenesis with XY women phenotype (46, XY DSD). In some others mammals, DMRT1 expression was shown to precede SOX9 up-regulation like in XX sex-reversed goats (FOXL2-/-) or in wild type XY rabbits where SRY and DMRT1 are both expressed in XY genital ridges a few days before testis cords formation. We thus assumed that DMRT1 was involved in sex determination in non-murine species, by directly targeting SOX9 expression. Thanks to CRISPR/Cas9 technology, we generated DMRT1 mutant rabbits. We show that the absence of DMRT1 leads to male-to-female sex reversal in XY homozygous mutants. At 20 days post conception, few days after sex-determination, XY DMRT1-/- gonads are devoid of testis cords structures but rather present a typical ovarian organization with cortical germ cells. Molecular studies confirmed the expressional up-regulation of ovary-driving genes (RSPO1, WNT4, FOXL2 and CYP19A1) and the downregulation of testis-specific ones (SOX9, SOX10 and DHH) while SRY expression is highly maintained in the developing XY ovary (RNA-sequencing and RT-qPCR data). Interestingly, some somatic cells of the XY DMRT1-/- gonads still present SOX9 protein expression. Nevertheless, this is not sufficient to trigger its target genes expression (e.g. AMH) in the developing XY ovary. Our data clearly involved DMRT1 as a key player in sex determination in rabbits. DMRT1 is required to SRY action on SOX9 expression and may also be necessary to SOX9 function on its own target genes. Interestingly, DMRT1 was recently shown to act as a pioneer factor, opening the chromatin and favouring the action of SOX protein such as SOX9. Investigation on DMRT1 targets and epigenetic mark (H3K4me1, H3K27ac and H3K27me3) deposition on these targets are in progress at early stages of rabbit gonadal differentiation.