The effects of perfluorononanoic acid (PFNA) on the immune system and its mechanism of action in mice have not been elucidated. Thus, BALB/c mice were exposed to the PFNA (0, 1, 3, or 5 mg/kg/day) for fourteen days. Exposure to PFNA led to a decrease in body weight and in the weight of the lymphoid organs. Cell cycle arrest and apoptosis were observed in the spleen and thymus following PFNA exposure. In the thymus, PFNA mostly modulated CD4+CD8+ thymocytes, whereas the F4/80+, CD11c+, and CD49b+ cells were major targets in the spleen. Although concanavalin A-induced T lymphocyte blastogenesis was not altered by PFNA, production of interleukin (IL)-4 and interferon-gamma by splenic lymphocytes was remarkably impaired. The levels of cortisol and adrenocorticotrophic hormone in sera were increased; however, the expression of glucocorticoid receptor in the thymus was unchanged. In addition, expression of the peroxisome proliferator-activated receptors (PPAR-alpha and PPAR-gamma) and IL-1beta were upregulated significantly in the thymus at a dose of 1 mg PFNA/kg/day. No significant changes in expression of the inhibitory protein IkappaBalpha and IkappaBalpha kinase were observed. Together, these results suggest that PFNA exerts toxic effects on lymphoid organs and T cell and innate immune cell homeostasis in mice and that these effects may result from the activation of PPAR-alpha, PPAR-gamma, and the hypothalamic-pituitary-adrenal axis. Interestingly, at the transcriptional level, the nuclear factor-kappa B signaling pathway appears to be uninvolved in the immunotoxic potential of PFNA.