The ATP binding cassette (ABC) ABCA3 gene encodes a lipid transporter critical for surfactant function at birth. To identify transcription factors that regulate ABCA3 expression in the lung, we identified by bioinformatic and functional analyses, two positive regulatory regions, located between base pairs (bp) -2591 to -1102 and bp -1102 to +11, relative to the exon 1 of the Abca3 gene promoter. The distal cassette contains consensus sequences predicting binding to lung transcription factors including FOXA2, C/EBP, GATA6, TTF-1 (Nkx2.), and NFATc3. The activity of the distal region from bp -2591 to -1102 was assessed in HeLa and MLE-15 cells. FOXA2, C/EBP, GATA6, TTF-1, and NFATc3 increased the activity of the Abca3-luciferase construct in a dose-dependent manner. The distal cassette conferred activation by FOXA2, C/EBP, GATA6, TTF-1, and NFATc3 in a position- and orientation-independent manner, serving as an enhancer-like regulatory element. The proximal Abca3 promoter region contained multiple sterol responsive element (SRE) binding sites. SRE binding protein (SREBP) 1c significantly increased the activity of the Abca3-luciferase construct in a dose-dependent manner, whereas SREBP-1a and SREBP-2 did not influence the Abca3 promoter activity. ChIP analyses demonstrated the binding of SREBP-1c, C/EBP, and TTF-1 to their respective regulatory elements. Conditional deletion of SREBP cleavage-activating protein, Scap, in respiratory epithelial cells in the mouse lung in vivo, inhibited the expression of SREBPs in concert with Abca3. Abca3 gene expression is mediated by discrete cis-acting cassettes that mediate pulmonary cell and lipid sensitive pathways regulating surfactant homeostasis.