The onset of steroidogenesis in human fetal testes (HFT) during the first trimester is poorly investigated. One important unresolved question is the ontogeny of steroidogenic enzymes and formation of steroidogenic pathways in the HFT at early pregnancy. Our aim was to explore steroidogenesis, the expression of steroidogenic enzymes and their maturation in the HFT at gestational weeks (GW) 8-12. Steroids in the HFT were analyzed by gas chromatography/coupled to tandem mass spectrometry. The expression of steroidogenic enzymes in the HFT at GW8-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that the HFT at GW8-9 produced low level of testosterone via the Delta(4) pathway and progesterone was the major steroid found in the testicular tissue. In contrast, more mature Leydig cells from the HFT at GW11-12 synthesized high levels of androgens via the Delta(5) pathway. We also observed a significant upregulation of the expression of StAR, CYP11A1, CYP17A1 and its accessory proteins, P450 oxidoreductase (POR) and cytochrome b5 in the HFT at GW11-12 compared to GW89. Altogether, our data suggest that that human fetal Leydig cells differentiate rapidly at the end of the first trimester by acquiring capacity to express high levels of steroidogenic enzymes and switch from the Delta(4) to the Delta(5) pathways to synthesize high levels of androgens due to maturation of the CYP17-POR-b5 complex.