Serum cortisol levels were evaluated in mice following intravenous administration of purified mycobacterial glycolipid trehalose 6,6′-dimycolate (TDM). C57BL/6 mice develop lung granulomas in response to TDM, while A/J mice are deficient in this process. Administration of TDM to C57BL/6 mice led to a rapid reduction in serum cortisol, concurrent with initiation of the granulomatous response and cytokine and chemokine mRNA induction. Cortisol levels were lowest on day 5 after TDM administration, but there was significant production of IL-6, TNF-α and IL-1β messages. Granuloma formation and full immune responsiveness to TDM were only apparent upon a sufficient decrease in levels of systemic cortisol. Treatment of the C57BL/6 mice with hydrocortisone abolished inflammatory responses. Histologically nonresponding A/J mice exhibited higher constitutive serum cortisol and demonstrated different kinetics of cortisol reduction upon administration of TDM. A/J mice demonstrated hyperplastic morphology in the suprarenal gland with a high degree of vacuolization in the medullary region and activation of cells in the zona fasciculata and zona reticularis. The A/J mice were dysregulated with respect to cytokine responses thought to be necessary during granuloma formation. The high constitutive serum cortisol in the A/J mice may therefore contribute to pulmonary immunoresponsiveness and the establishment of an environment counterproductive to the initiation of granulomatous responses. The identification of a mycobacterial glycolipid able to influence serum cortisol levels is unique and is discussed in relation to immunopathology during tuberculosis disease.