Aldosterone synthase (CYP11B2) is responsible for the final step in aldosterone synthesis and is importantly regulated by angiotensin-II (Ang II) through diverse pathways. However, under pathological conditions, such as in hyperaldosteronism, the regulation becomes disordered. The transcription factor steroidogenic factor-1 (SF-1) is important in regulating the endocrine system and is overexpressed in aldosterone-producing adenoma (APA), a common cause of hyperaldosteronism. Overexpression of SF-1 has been extensively studied, but little in-depth information is available regarding the effects of inhibitory SF-1 on CYP11B2 and Ang II. In this paper, we have investigated the roles of down-regulated SF-1 in basal and Ang II-induced CYP11B2 expression using SF-1-specific short hairpin RNA. Inhibitory SF-1 was found to decrease the sensitivity of CYP11B2 and aldosterone to Ang II stimulation, whereas a down-regulation of SF-1 enhanced basal CYP11B2 expression and aldosterone production in H295R cells. Considering these differential effects of SF-1 on aldosterone production, these results might provide a new insight into the understanding of hyperaldosteronism.