The hypothalamic-pituitary-adrenal axis is the primary neuroendocrine system activated to re-establish homeostasis during periods of stress, including critical illness and major surgery. During critical illness, evidence suggests that locally induced inflammation of the adrenal gland could facilitate immune-adrenal cross-talk and, in turn, modulate cortisol secretion. It has been hypothesized that immune cells are necessary to mediate the effect of inflammatory stimuli on the steroidogenic pathway that has been observed in vivo. To test this hypothesis, we developed and characterized a trans-well co-culture model of THP1 (human monocytic cell)-derived macrophages and ATC7 murine zona fasciculata adrenocortical cells. We found that co-culture of ATC7 and THP1 cells results in a significant increase in the basal levels of IL-6 mRNA in ATC7 cells, and this effect was potentiated by treatment with LPS. Addition of LPS to co-cultures of ATC7 and THP1 significantly decreased the expression of key adrenal steroidogenic enzymes (including StAR and DAX-1), and this was also found in ATC7 cells treated with pro-inflammatory cytokines. Moreover, 24-h treatment with the synthetic glucocorticoid dexamethasone prevented the effects of LPS stimulation on IL-6, StAR and DAX-1 mRNA in ATC7 cells co-cultured with THP1 cells. Our data suggest that the expression of IL-6 and steroidogenic genes in response to LPS depends on the activation of intra-adrenal immune cells. Moreover, we also show that the effects of LPS can be modulated by glucocorticoids in a time- and dose-dependent manner with potential implications for clinical practice.