Background This study sought to determine whether binding of acetylated C/EBPβ to α-SMA promoter could affect its activity and was essential for EMT and extracellular matrix deposition in IPF using in vitro model. Methods Through western blotting, the expression of EMT and C/EBPβ were detected in A549 cells with TGF-β as pulmonary fibrotic model in vitro. Moreover, the expression of C/EBPβ mRNA via Real Time-PCR and Collagen-I expression using ELISA were performed. The luciferase activity assay was used to examine the activity of C/EBPβ. The knockdown expression of C/EBPβ gene was prepared in A549 cells with C/EBPβ siRNA. We also investigated the effect of deacetylation of C/EBPβ on EMT using SIRT1. The binding ability of C/EBPβ with the α-SMA promoter was affirmed via ChIP and EMSA. Furthermore, the relationship between α-SMA and acetylated C/EBPβ was investigated using the co-immunoprecipitation. Results SiRNA-mediated knockdown of C/EBPβ in A549 cells attenuated TGF-β1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPβ and the α-SMA promoter was dynamically monitored. Furthermore, it was confirmed that deacetylation of C/EBPβ in A549 cells successfully ameliorated TGF-β1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. Conclusions Collectively, our data suggested that the EMT and fibrotic effect of TGF-β1 could be dependent on acetylated C/EBPβ-mediated regulation of α-SMA gene activity. This thus suggests that C/EBPβ acetylation may play a central role in pulmonary fibrosis.