Steroidogenesis from cholesterol in placental trophoblasts is fundamentally involved in the establishment and maintenance of pregnancy. The transcription factor gene Heart And Neural crest Derivatives expressed 1 (Hand1)promotes differentiation of mouse trophoblast giant cells. However, the role of HAND1in human trophoblasts remains unknown. Here, we report that HAND1 inhibits human trophoblastic progesterone (P4) and estradiol (E2) from cholesterol through down-regulation of the expression of steroidogenic enzymes including aromatase, P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1). Mechanically, while HAND1 inhibits transcription of aromataseby directly binding to aromatasegene promoter, it restrains transcription of P450sccby up-regulation of the methylation status of P450sccgene promoter through its binding to ALKBH1, a demethylase. Unlike aromatase and P450scc, HAND1 decreases 3β-HSD1mRNA levels by reduction of its RNA stability through binding to and subsequent destabilization of protein HuR. Finally, HAND1 suppresses circulating P4 and E2 levels derived from JEG-3 xenograft, and attenuates uterine response to P4 and E2. Thus, our results uncover a hitherto uncharacterized role of HAND1 in regulation of cholesterol metabolism in human trophoblasts, which may help pinpoint the underlying mechanisms involved in supporting the development and physiological function of the human placenta.