Pulmonary fibrosis is characterized by the excessive deposition of a collagen-rich extracellular matrix. The accumulation of collagen within the lung interstitium leads to impaired respiratory function. Furthermore, smooth muscle actin-positive myofibroblasts within the fibrotic lung contribute to disease progression. Because collagen and smooth muscle cell α-actin are coordinately expressed in the setting of fibrosis, the hypothesis was tested that specific transcriptional regulators of the myocardin family might also regulate collagen gene expression in myofibroblasts. Myocardin-related transcription factors (MRTFs), through their interaction with the serum-response factor (SRF) on CArG box regulatory elements (CC(A/T)6GG), are important regulators of myofibroblast differentiation. MRTF-A transactivated type I collagen gene reporters as much as 100-fold in lung myofibroblasts. Loss of functional MRTF-A using either a dominant negative MRTF-A isoform, shRNA targeting MRTF-A, or genetic deletion of MRTF-A in lung fibroblasts significantly disrupted type I collagen synthesis relative to controls. Analysis of the COL1A2 proximal promoter revealed a noncanonical CArG box (CCAAACTTGG), flanked by several Sp1 sites important for MRTF-A activation. Chromatin immunoprecipitation experiments confirmed the co-localization of MRTF-A, SRF, and Sp1 bound to the same region of the COL1A2 promoter. Mutagenesis of either the noncanonical CArG box or the Sp1 sites significantly disrupted MRTF-A activation of COL1A2. Together, our findings show that MRTF-A is an important regulator of collagen synthesis in lung fibroblasts and exhibits a dependence on both SRF and Sp1 function to enhance collagen expression.