Fibroblasts migrating to peritoneum injuries play an important role in the development of postoperative peritoneal adhesions due to the excessive synthesis and deposition of extracellular matrix (ECM). This effect is mainly induced by the transforming growth factor-β (TGF-β). Studies indicate that elevated TGF-β1 levels and TGF-β1/Smad signaling are both implicated in the formation of peritoneal adhesions. To confirm the effect of TGF-β1/Smad signaling interference in regulating excessive ECM synthesis, a total of four different R-Smad-targeting small interference RNA (siRNA) duplexes (Smad2-500, Smad2-956, Smad3-378, Smad3-1385) were tested in this study using a TGF-β1-stimulated adhesion tissue fibroblasts (ATFs) cell model. The in vitro assessments show that all proposed siRNAs are capable of significantly downregulating the mRNA and protein levels of Smad2 and Smad3 in ATFs. They also inhibit the phosphorylation of both Smads, which confirms their effect in blocking the TGF-β1/Smad signaling pathway. Moreover, the siRNA duplexes can appreciably lower the elevated levels of fibronectin and collagen 3 alpha 1 (COL3A1) in TGF-β1-stimulated ATFs, and the Smad3-378 siRNA can actually restore both molecules (fibronectin and COL3A1) to normal levels. Therefore, the Smad3-378 siRNA is suitable for both adhesion prevention and wound healing. Overall, our results indicate that postoperative adhesion prophylaxis may be achieved by temporarily blocking TGF-β1/Smad signaling transduction.