There is considerable evidence supporting the role of calcium signaling in adrenal regulation of both aldosterone synthase (CYP11B2) and aldosterone production. However, there have been no studies that investigated the role played by the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) in adrenal cells. In this study we investigated the role of CaMKK in adrenal cell aldosterone production. To determine the role of CaMKK, we used a selective CaMKK inhibitor (STO-609) in the HAC15 human adrenal cell line. Cells were treated with angiotensin II (Ang II) or K+ and evaluated for the expression of steroidogenic acute regulatory protein and CYP11B2 (mRNA/protein) as well as aldosterone production. We also transduced HAC15 cells with lentiviral short hairpin RNAs of CaMKK1 and CaMKK2 to determine which CaMKK plays a more important role in adrenal cell regulation of the calcium signaling cascade. The CaMKK inhibitor, STO-609, decreased aldosterone production in cells treated with Ang II or K+ in a dose-dependent manner. STO-609 (20 μM) also inhibited steroidogenic acute regulatory protein and CYP11B2 mRNA/protein induction. CaMKK2 knockdown cells showed significant reduction of CYP11B2 mRNA induction and aldosterone production in cells treated with Ang II, although there was no obvious effect in CaMKK1 knockdown cells. In immunohistochemical analysis, CaMKK2 protein was highly expressed in human adrenal zona glomerulosa with lower expression in the zona fasciculata. In conclusion, the present study suggests that CaMKK2 plays a pivotal role in the calcium signaling cascade regulating adrenal aldosterone production.