5α-Androstan-3β-ol, a steroidal androgen, demonstrates selective interaction with cellular components, showing affinity for the androgen receptor (AR) with an IC50 of 1.7 µM and activating the pregnane X receptor (PXR) in HepG2 cells with an EC50 of 0.8 µM. It selectively inhibits the hydroxylase activities of 16α- and 6β-hydroxylase over 16β- or 7α-hydroxylase, with IC50 values of 70 and 110 µM, compared to >150 µM for the latter enzymes, indicating a specificity in its enzyme inhibition profile. Additionally, administration of this compound at a dosage of 30 mg/kg results in a decrease in testis weight and sperm production in mice, highlighting its biological effects on male reproductive parameters.