OBJECTIVE: To report a novel case of semen cryopreservation after testicular sperm extraction in an adolescent transgender female without cessation of gonadotropin-releasing hormone (GnRH) agonist therapy and feminizing hormone therapy. METHODS: This is a case report of a 16-year-old transgender female using leuprolide acetate for 4 years and estradiol for 3 years requesting semen cryopreservation at the time of gender-affirming orchiectomy. She desired to proceed without cessation of gender affirming hormone therapy. The patients consent was obtained for written publication. RESULTS: The patient underwent testicular sperm extraction followed by orchiectomy. The sample was processed and cryopreserved in a 1:1 Test Yolk Buffer. Multiple early and late spermatids were identified as well as spermatagonium in the TESE specimen. CONCLUSIONS: Advanced spermatogenesis may occur in the presence of a GnRH agonist. Cessation of GnRH agonist therapy may not be essential for semen cryopreservation in adolescent transgender females.