AbstractPreterm birth is characterized by severe lung immaturity that is frequently treated antenatally or postnatally with the synthetic steroid betamethasone. The underlying cellular targets and pathways stimulated by betamethasone in the fetal lung are poorly defined. In this study, betamethasone was compared with corticosterone in steroid-treated primary cultures of fetal rat lung fibroblasts stimulated for 6 hours and analyzed by whole-cell transcriptome sequencing and glucocorticoid (GC) receptor (GR) chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. Strikingly, betamethasone stimulated a much stronger transcriptional response compared with corticosterone for both induced and repressed genes. A total of 483 genes were significantly stimulated by betamethasone or corticosterone, with 476 stimulated by both steroids, indicating a strong overlap in regulation. Changes in mRNA levels were confirmed by quantitative PCR for eight induced and repressed target genes. Pathway analysis identified cell proliferation and cytoskeletal/cell matrix remodeling pathways as key processes regulated by both steroids. One target, transglutaminase 2 (Tgm2), was localized to fetal lung mesenchymal cells. Tgm2 mRNA and protein levels were strongly increased in fibroblasts by both steroids. Whole-genome GR ChIP-Seq analysis with betamethasone identified GC response element–binding sites close to the previously characterized GR target genes Per1, Dusp1, Fkbp5, and Sgk1 and near the genes identified by transcriptome sequencing encoding Crispld2, Tgm2, Hif3α, and Kdr, defining direct genomic induction of expression in fetal lung fibroblasts via the GR. These results demonstrate that betamethasone stimulates specific genes and cellular pathways controlling cell proliferation and extracellular matrix remodeling in lung mesenchymal fibroblasts, providing a basis for betamethasone’s treatment efficacy in preterm birth.