<p>ATP Binding Cassette Subfamily C Member 3 (ABCC3) is a membrane transporter that exports diverse compounds, influencing drug resistance in cancers and impacting metabolic and malignant diseases. We previously reported <i>ABCC3</i> upregulation in human adrenal cells under lipid peroxidation-induced oxidative stress. We investigated ABCC3 expression and function in the human adrenal cortex in the context of primary aldosteronism, focusing on its relationship to CYP11B2 (aldosterone synthase) in zona glomerulosa (ZG) cells and aldosterone-producing lesions. ABCC3 expression in aldosterone-producing adenomas (APAs) was markedly reduced compared to the adjacent adrenal cortex, cortisol-producing adenomas, and non-functioning adenomas. The reduction in APAs showed genotypic variability: in APAs harboring KCNJ5 mutations—known drivers of autonomous aldosterone production via altered potassium channel function—ABCC3 mRNA/protein levels were significantly higher than in wild-type KCNJ5 APAs. Spatial transcriptomics and immunofluorescence colocalization revealed an inverse expression pattern between ABCC3 and CYP11B2 (aldosterone synthase) in APAs and aldosterone-producing micronodules (APMs). Notably, high ABCC3 expression was exclusively observed in adrenal ZG cells that lacked CYP11B2 expression. This expression pattern distinguishes ABCC3 from common ZG markers such as KCNJ5 and DAB2, which are expressed across adrenal ZG cells, APMs, and APA tumor cells. In cultured human adrenocortical cells, both angiotensin II stimulation and induced expression of the KCNJ5-L168R mutation resulted in upregulation of <i>CYP11B2</i> transcription while concomitantly suppressing <i>ABCC3</i> expression. In conclusion, ABCC3 serves as a novel marker of CYP11B2-negative ZG cells, providing a histopathological tool to differentiate normal adrenal zonation from aldosterone-producing lesions in primary aldosteronism, as well as from cortisol-producing and nonfunctional adenomas.</p>