Background: MicroRNAs are a group of small RNAs that regulate gene expression at the posttranscriptional level. They regulate almost every aspect of cellular processes. In this study, we investigated whether miR-27b regulates pulmonary fibroblast activation. Results: We found that miR-27b was down-regulated in fibrotic lungs and fibroblasts from an experimental mouse model of pulmonary fibrosis. The overexpression of miR-27b with a lentiviral vector inhibited TGF beta 1-stimulated mRNA expression of collagens (COL1A1, COL3A1, and COL4A1) and alpha-smooth muscle actin, and protein expression of Col3A1 and alpha-smooth muscle actin in LL29 human pulmonary fibroblasts. miR-27b also reduced contractile activity of LL29. TGF beta receptor 1 and SMAD2 were identified as the targets of miR-27b by 3'-untranslated region luciferase reporter and western blotting assays. Conclusions: Our results suggest that miR-27b is an anti-fibrotic microRNA that inhibits fibroblast activation by targeting TGF receptor 1 and SMAD2. This discovery may provide new targets for therapeutic interventions of idiopathic pulmonary fibrosis.