The Clara cell secretory protein (CCSP, also CC-10/uterglobin) is a 16-kD homodimeric protein abundantly expressed in the airways of mammals. Although the molecular function is unknown, gene-targeting studies indicate CCSP as a regulator of lung inflammation following acute respiratory infection or injury. CCSP is decreased in the lungs of mice following acute Pseudomonas aeruginosa (P.a.) infection. In the present study, the role of decreased promoter function in the regulation of CCSP by P.a. was assessed using an in vitro co-culture system and in vivo studies of transgenic mice. CCSP promoter activity in lung epithelial cells was markedly decreased by P.a. or tumor necrosis factor-α (TNF-α) in a dose-dependent manner. Regulation of CCSP promoter function by either P.a. or TNF-α was localized to the proximal 166 bp flanking region of the CCSP promoter activity. Decreased regulation of the CCSP promoter by P.a. or TNF-α was specific to CCSP, as human surfactant protein D (SP-D) promoter activity was unaffected or increased by P.a. or TNF-α, respectively. A neutralizing antibody against human TNF-α was able to reverse both the TNF-α-mediated as well as P.a.-mediated decrease in CCSP promoter function in lung epithelial cells. TNF-α secretion by lung epithelial cells coincided with the decrease in CCSP promoter function following P.a. administration. Using a transgenic mouse model, P.a. administration to the lung markedly attenuated CCSP promoter-conferred gene expression in vivo. The attenuation of CCSP promoter activity in lung epithelial cells by P.a. involves, in part, autocrine/paracrine secretion of TNF-α, which in turn regulates CCSP transcription through cis-active elements in the proximal promoter region.