Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) in the lung parenchyma. The abnormal ECM deposition slowly overtakes normal lung tissue, disturbing gas exchange and leading to respiratory failure and death. ECM cross-linking and subsequent stiffening is thought to be a major contributor of disease progression and also promotes the activation of transforming growth factor (TGF)-β1, one of the main profibrotic growth factors. Lysyl oxidase-like (LOXL) 1 belongs to the cross-linking enzyme family and has been shown to be up-regulated in active fibrotic regions of bleomycin-treated mice and patients with IPF. We demonstrate in this study that LOXL1-deficient mice are protected from experimental lung fibrosis induced by overexpression of TGF-β1 using adenoviral (Ad) gene transfer (AdTGF-β1). The lack of LOXL1 prevented accumulation of insoluble cross-linked collagen in the lungs, and therefore limited lung stiffness after AdTGF-β1. In addition, we applied mechanical stretch to lung slices from LOXL1+/+ and LOXL1−/− mice treated with AdTGF-β1. Lung stiffness (Young's modulus) of LOXL1−/− lung slices was significantly lower compared with LOXL1+/+ lung slices. Moreover, the release of activated TGF-β1 after mechanical stretch was significantly lower in LOXL1−/− mice compared with LOXL1+/+ mice after AdTGF-β1. These data support the concept that cross-linking enzyme inhibition represents an interesting therapeutic target for drug development in IPF.