Objectives Describe the effect of 50 mcg vaginal 17-β-estradiol (E2) cream on vaginal maturation, serum estrogen levels, atrophic symptoms, and biomarkers of oxidative stress and tissue remodeling in postmenopausal women without prolapse. Methods Seventeen women, 65 years or older, applied intravaginal E2 cream nightly for eight weeks, then twice weekly for eight weeks. Vaginal biopsies, serial blood draws, and atrophic symptoms were obtained at baseline, eight, and sixteen weeks. Changes in atrophic symptoms, vaginal maturation indices (VMI), and serum E2 were measured. Immunohistochemical staining characterized levels of transforming growth factor-beta (TGF-β), nuclear factor kappa B (NFKB), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and thrombospondin (TSP). Results Serum E2 levels (pg/ml) were unchanged from baseline (mean (SD)) 7.7 (3.3) to eight 9.7 (5.7) and sixteen 8.7 (5.8) (p = 0.24) weeks. VMI (mean (SD)) improved from baseline 34.2 (18.3) to eight 56.7 (13.1) and sixteen 54.5 (11.3) (p < 0.001) weeks with no difference between eight and sixteen weeks. Vaginal dryness (p = 0.03) and itching (p = 0.02) improved. Tissue biomarker levels did not change (TGF-β p = 0.35, NFKB p = 0.74, eNOS p = 0.80, iNOS p = 0.24, TSP p = 0.80). Discussion Vaginal E2 improved atrophic symptoms and VMI without elevating serum E2. Tissue remodeling biomarkers did not change.