Objective To investigate the effect of docosahexaenoic acid (DHA) in the blood and tissues of patients with primary aldosteronism (PA) on aldosterone synthesis in adrenocortical cells and its potential targets of action. Methods Sixty patients with unilateral and bilateral PA were selected. The concentration of DHA in peripheral blood of the patients and in pathologic tissues after surgery for unilateral PA was measured using mass spectrometry. HAC15 adrenocortical cells were used as an in vitro model. The effects of different concentrations of DHA (0, 20, 80, and 200 μM) on the cell proliferation activity of CCK-8 was compared. The aldosterone secretion, and the changes of protein and mRNA expression of CYP11B2 were analyzed. The effect of DHA on calcium signaling was analyzed using calcium ion fluorescence assay. Results Peripheral blood DHA levels were higher in patients with unilateral PA than those in patients with bilateral PA. Histopathology of patients with aldosterone adenoma ( APA) showed higher tumor DHA levels than those of the peripheral cortex. The DHA drug had an inhibitory effect on the proliferation of HAC15 cells with a significant decrease in the rate of cell proliferation (P<0. 05) but did not significantly induce apoptosis. Aldosterone secretion was significantly higher in HAC15 cells after DHA intervention (P<0. 001). The protein and mRNA of CYP11B2 were up-regulated, and the intracellular calcium ion level was also significantly elevated (P<0. 001). Conclusions DHA may promote the expression of CYP11B2 to indorse the synthesis and secretion of HAC15 aldosterone by regulating the calcium signaling pathway.