The immunomodulatory potential of thymulin and zinc (Zn2+) in the perinatal alveolar epithelium is not well characterized. In an in vitro model of fetal alveolar type II epithelial cells (FATEII), we have investigated the exhibition of an anti-inflammatory activity of this peptide hormone. Thymulin selectively ameliorated, in a dose-dependent manner, the endotoxin (ET/LPS [lipopolysaccharide])-induced release of IL-1β, but not IL-6 or TNF-α. Furthermore, Zn2+, an anti-inflammatory antioxidant, which is required for the biological activity of thymulin, independently reduced the secretion of IL-1β, TNF-α and, to a lesser extent, at a supraphysiologic dose (1mM), IL-6. The underlying cellular and molecular pathways associated with the anti-inflammatory effect of thymulin and Zn2+ in the alveolar epithelium are not well established. Further in this study, the role of cyclic AMP (cAMP) in the anti-inflammatory effect of thymulin was investigated, in addition to unraveling the possible involvement of the NF-κB pathway. Interestingly, thymulin upregulated, in a dose- and time-dependent manner, the release of the nucleotide cAMP. To understand whether the inhibitory effect of thymulin on cytokine release is cAMP-dependent, Forskolin, a labdane diterpene known to elevate intracellular cAMP, was shown to reduce the secretion of IL-1β and TNF-α, but not IL-6, an effect mimicked by dibutyryl-cAMP (dbcAMP), an analog of cAMP. Alveolar epithelial cells treated with thymulin markedly showed a downregulation of the nuclear translocation of RelA (p65), the major transactivating member of the NF-κB family, in addition to NF-κB1 (p50) and c-Rel (p75), an effect mildly substantiated with Zn2+. Furthermore, thymulin/Zn2+ reduced, in a dose-dependent manner, the DNA-binding activity of NF-κB (RelA/p65). These results indicate that the anti-inflammatory effect of thymulin, which is mediated by cAMP, is NF-κB-dependent and involves the downregulation of the release of proinflammatory cytokines, particularly IL-1β, an effect synergistically amplified, at least in part, by Zn2+. The molecular regulation of thymulin via a NF-κB-dependent pathway is critical to understanding the anti-inflammatory alleviating role of this nonapeptide in regulating proinflammatory signals.