Until recently, mammalian sex differentiation was thought to be finalized in the embryo. Development of the tubular genital tract, external genitalia, secondary sexual characteristics and sexual behavior are determined largely by the developing gonad. In the last decade, however, it has been shown that continuous sex maintenance is required throughout life. We document proposed ovary to testis transdifferentiation in six captive South African lionesses. These lions were phenotypically female until, as older adults, they began to develop manes, clitoromegaly and increased levels of restlessness and aggression. Similar phenotypic and behavioral changes have been reported very rarely in free-ranging lionesses. Five of the lionesses had been treated repeatedly with deslorelin until they stopped showing estrus. Serum levels of testosterone were similar to those recorded in domestic tom cats in the three lionesses tested. Histological examination revealed an absence of ovarian follicles and small to large scattered islands of interstitial-like cells associated, in larger lesions, with Sertoli-like cells arranged in tubular structures. Oviduct and uterus were present in the four animals in which these were sampled. To date, most studies on this phenomenon have been conducted in laboratory animals. This is the first report of this condition in felids. •Six lionizers with manes had testicular-like cells but no follicles in their ovaries.•Masculinization regressed after ovariohysterectomy.•Germ cell death due to contraception may result in ovarian transdifferentiation.