Dietary supplements used by recreational and elite athletes for performance enhancement might contain undeclared, unlawfully added ingredients. One of those ingredients is 5α-hydroxy-laxogenin, which is sold in dietary supplements marketed as a natural compound with anabolic effects. It has been shown that 5α-hydroxy-laxogenin is not naturally occurring, but rather of synthetic origin. Previously, we observed that 5α-hydroxy-laxogenin can bind to and activate the androgen receptor in a cell-based bioassay. To investigate its androgenic potential in vivo, we treated orchiectomized rats with three different dosages of 5α-hydroxy-laxogenin for 2 weeks. Effects were neither observed on the wet weights of the androgen target tissues prostate, seminal vesicle or penis nor on the wet weights of the anabolic target tissue musculus levator ani or on skeletal hindlimb muscles. Au contraire, significantly higher atrophy was seen for some of the target tissues in the animals treated with the highest 5α-hydroxy-laxogenin dosage (36 mg/kg bw). While in silico docking supports the androgen receptor binding previously observed in vitro, we observed neither androgenic nor anabolic effects of 5α-hydroxy-laxogenin in vivo in castrated male rats.