Fibrosis of the subsynovial connective tissue (SSCT) is a characteristic finding in patients with idiopathic carpal tunnel syndrome (CTS). Idiopathic CTS frequently occurs in postmenopausal women; therefore, female steroid hormones, especially estrogens, may be involved in its development. In this study, we evaluated the effect of the estradiol on the expression of genes and proteins related to fibrosis of SSCT fibroblasts from patients with idiopathic CTS. This study included 10 postmenopausal women (mean age 76 years). Fibroblasts derived from SSCT were treated with estradiol (10 -10 M), and the expression levels of TGF-β-responsive genes were evaluated. The relationships between the expression of untreated estrogen receptor α (ERα) and ERβ and changes in gene expression due to estradiol treatment were examined by quantitative real-time polymerase chain reaction. The effects of 10 M estradiol on collagen type I (Col1) and collagen type III (Col3) protein expression levels were also evaluated by fluorescent staining. The relationships between ERα/β and Col1/3 expression were evaluated by immunohistochemical staining. The reduction in Col1A1 mRNA expression due to estradiol treatment was positively correlated with ERα expression (r = 0.903, p < 0.01). At the protein level, expression of Col1 and Col3 were down-regulated. These results indicated that ERα-mediated signaling may be involved in the regulation of Col1A1, and its regulatory effect may be dependent on the ERα expression level. The accurate evaluation of ERα expression level in the SSCT of individual patients with idiopathic CTS might guide the effective use of new estrogen replacement therapy.