Pleural dissemination is commonly associated with metastatic advanced lung cancer. The injury of pleural mesothelial cells (PMCs) by soluble factors, such as transforming growth factor-beta1 (TGF-β1), is a major driver of lung cancer pleural dissemination (LCPD). In this study, we examine the effects of TGF-β1 on PMC injury and the ability of TGF-β1 inhibition to alleviate this effect both in vitro and in vivo. PMCs were co-cultured with the high TGF-β1-expressing lung cancer cell line A549 and with various TGF-β1 signaling inhibitors. Expression of cleaved-caspase 3, cleaved-caspase 9, p21, and p16 were evaluated by Western blot and immunofluorescent confocal imaging. Apoptosis was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltrazoliumbromide assay and AnnexinV-propidium iodide (PI) staining. PMC senescence was assessed by staining for senescence-associated β-galactosidase (SA-β-Gal). The ability of lung cancer cells (LCCs) to adhere to injured PMCs was investigated using an LCC-PMC adhesion assay. In our mouse model, PMC injury status was monitored by hematoxylin-eosin (H&E) and Masson's trichrome staining. LCCs expressing high levels of TGF-β1 induce apoptosis and senescence of PMCs in a co-culture system. Injured PMCs adhere to LCCs, which may further promote LCPD. Importantly, PMC monolayer injury could be reversed with TGF-β1 inhibitors. This was consistent with our in vivo data showing that the TGF-β1 inhibitor SB-431542 attenuated PMC barrier injury induced by A549 culture medium in our mouse model. Our study highlights the importance of TGF-β1 signaling in LCPD and establishes this signaling pathway as a potential therapeutic target in the disease.