Fibroblast-to-myofibroblast differentiation is one of the most important characteristics of pulmonary fibrosis, and screening natural compounds targeting fibroblast differentiation is always a promising approach to discover drug candidates for treatment of pulmonary fibrosis. Trehalose reportedly has many potential medical applications, especially in treating neurodegeneration diseases. However, it remains unclear whether trehalose suppresses lung fibroblast differentiation. In this work, we found that trehalose decreased the expression levels of α-smooth muscle actin (α-SMA) following the induction of transforming growth factor β1 (TGF-β1) in pretreatment, co-treatment, and post-treatment groups. Trehalose also reduced the production of type I collagen, lung fibroblast-containing gel contractility and cell filament formation in TGF-β1-stimulated MRC-5 cells. Although trehalose is a known autophagy inducer, our results showed that its suppressive effect on fibroblast differentiation was not via trehalose-induced autophagy. And it did not affect canonical TGFβ/Smad2/3 pathway. By applying proteomic profiling technology, we demonstrated that the downregulation of β-catenin was involved in the trehalose-repressive action on fibroblast differentiation. The β-catenin agonist, SKL2001, reversed the suppressive effect of trehalose on fibroblast differentiation. Overall, these experiments demonstrated that trehalose suppressed fibroblast differentiation via the downregulation of β-catenin, but not through canonical autophagy and TGFβ/Smad2/3 pathway, which is not only a novel understanding of trehalose, but also quite helpful for in vivo research of trehalose on pulmonary fibrosis in future.