Background: Hypoxia is a critical physiological and pathological condition known to influence various cellular processes, including steroidogenesis. While previous studies, including our own, have highlighted the regulatory effects of Hypoxia-Inducible Factor 1α (HIF1α) on steroid production, the specific molecular mechanisms remain poorly understood. This study investigates the role of hypoxia and HIF1α in steroid biosynthesis across multiple experimental models during acute exposure to low oxygen levels. Methods: To assess the extent to which acute hypoxia modulates steroidogenesis, we employed several approaches, including the Y1 adrenocortical cell line, and a conditional HIF1α-deficient mouse line in the adrenal cortex. We focused on various regulatory patterns that may critically suppress steroidogenesis. Results: In Y1 cells, hypoxia upregulated specific microRNAs in a HIF1α-dependent manner, resulting in the suppression of mRNA levels of critical steroidogenic enzymes and a subsequent reduction in steroid hormone production. The hypoxia/HIF1α-dependent induction of these microRNAs and the consequent modulation of steroid production were confirmed in vivo. Notably, using our adrenocortical-specific HIF1α-deficient mouse line, we demonstrated that the increase in miRNA expression in vivo is also directly HIF1α-dependent, while the regulation of steroidogenic enzymes (e.g., StAR and Cyp11a1) and steroid production occurs at the level of protein translation, revealing an unexpected layer of control under hypoxic/HIF1 α conditions in vivo. Conclusions: These findings elucidate the molecular mechanisms underlying acute hypoxia/HIF1α-induced changes in steroid biosynthesis and may also be useful in developing new strategies for various steroid hormone pathologies.