Fibrosis of the subsynovial connective tissue (SSCT) in carpal tunnel syndrome (CTS) patients is increasingly recognized as an important aspect of CTS pathophysiology. In this study, we evaluated the effect of blocking profibrotic pathways in fibroblasts from the SSCT in CTS patients. Fibroblasts were stimulated with transforming growth factor β1 (TGF‐β1), and then treated either with a specific fibrosis pathway inhibitor targeting TGF‐β receptor type 1 (TβRI), platelet‐derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), or vascular endothelial growth factor receptor (VEGFR). Fibrosis array and quantitative real‐time polymerase chain reaction of fibrotic genes were evaluated. Array gene expression analysis revealed significant down‐regulation of multiple fibrotic genes after treatment with TβRI, PDGFR, and VEGFR inhibitors. No array fibrotic genes were significantly down‐regulated with EGFR inhibition. Further gene expression analysis of known CTS fibrosis markers collagen type I A2 (Col1), collagen type III A1 (Col3), connective tissue growth factor (CTGF), and SERPINE1 showed significantly down‐regulation after TβRI inhibition. In contrast, VEGFR inhibition significantly down‐regulated CTGF and SERPINE1, whereas, PDGFR and EGFR inhibition significantly down‐regulated Col3. Taken together the inhibition of TβRI appears to be the primary mediator of fibrotic gene expression in fibroblasts from CTS patients. TGF‐β/Smad activity was further evaluated, and as expected inhibition of Smad activity was significantly down‐regulated after inhibition of TβRI, but not with PDGFR, VEGFR, or EGFR inhibition. These results indicate that local therapies specifically targeting TGF‐β signaling alone or in combination offer the potential of a novel local antifibrosis therapy for patients with CTS. In this study, we evaluated the effect of blocking profibrotic pathways by receptor blocking agents in fibroblasts from patients with carpal tunnel syndrome (CTS). There was significant down‐regulation of multiple fibrotic genes after treatment with a TFFβ1R (SD208) inhibitor, with smaller effects from PDGFR (AG1296) and VEGFR (Axitinib) inhibitors. These results indicate that local therapies specifically targeting TGF‐β signaling, alone or in combination with other pathways, offer the potential of a novel local anti‐fibrosis therapy for patients with CTS.