Postoperative intestinal adhesions are common and serious complications after surgery that can cause pain and potential mortality. Our previous study confirmed that silkworm pupae carboxymethyl chitosan (SP-carboxymethyl chitosan) reduced postoperative adhesion in vivo. Here, we elucidated the inhibitory effects of SP-carboxymethyl chitosan on mouse L929 fibroblasts. Cells were exposed to SP-carboxymethyl chitosan for 72 h, then the inhibitory effects were assessed via transforming growth factor-β1 (TGF-β1)/Smads, plasminogen activator inhibitor 1 (PAI-1), and tissue-type plasminogen activator (t-PA) signaling. The results showed that SP-carboxymethyl chitosan suppressed cell hyperplasia and significantly attenuated the gene and protein expressions of the TGF-β1/Smads signaling pathways. We also confirmed that t-PA/PAI-1 greatly increased for all SP-carboxymethyl chitosan-treated groups compared to the control. These findings suggest that SP-carboxymethyl chitosan may affect L929 cell proliferation through the TGF-β1/Smads signaling pathway to prevent adhesion after an operation.