Type I collagen is a heterotrimeric extracellular matrix protein consisting of two α1(I) chains and one α2(I) chain. During liver fibrosis, activated hepatic stellate cells (HSCs) are the major source of the type I collagen that accumulates in the damaged tissue. Expression of α1(I) and α2(I) collagen mRNA is increased 60-fold compared with quiescent stellate cells and is due predominantly to post-transcriptional message regulation. Specifically, a stem-loop structure in the 5'-untranslated region of α1(I) collagen mRNA may regulate mRNA expression in activated HSCs through its interaction with stem-loop binding proteins. The stem-loop may also be necessary for efficient production and folding of the type I collagen heterotrimer. To assess the role of the stem-loop in type I collagen expression in vivo, we generated a knock-in mouse harboring a mutation that abolished the stem-loop structure. Heterozygous and homozygous knock-in mice exhibited a normal phenotype. However, steady-state levels of α1(I) collagen mRNA decreased significantly in homozygous mutant MEFs as well as HSCs; intracellular and secreted type I collagen protein levels also decreased. Homozygous mutant mice developed less liver fibrosis. These results confirm an important role of the 5' stem-loop in regulating type I collagen mRNA and protein expression and provide a mouse model for further study of collagen-associated diseases.