Aims: This study aims to assess vaginal wall angioarchitecture and function in women with vulvovaginal atrophy (VVA) and determine the effect of topical estrogen on the vaginal microcirculation. Materials and Methods: In this prospective observational study, incident dark field imaging was used to assess the vaginal microcirculation. In patients with VVA, measurements were performed before and after treatment with topical estrogen and compared to measurements performed in women without VVA. Vaginal angioarchitecture was studied by assessing microcirculatory architecture and capillary tortuosity scores at four regions of the vaginal wall. In addition, the capillary density and microvascular flow index (MFI) were obtained. Results: Seventeen women were included in this study. Of these, eight women were diagnosed with VVA and nine women were considered healthy controls. Significant differences were observed between groups with regard to microcirculatory architecture scores. The architecture of the microvasculature in women with VVA was characterized by the appearance of a vascular network without capillary loops, whereas an array of capillary loops was predominantly seen in women without VVA. After estrogen treatment, no difference in architecture scores between patients and healthy controls was observed. Capillary tortuosity, capillary density, and MFI were similar in both groups before and after estrogen treatment. Conclusions: The architecture of vaginal microvasculature is altered in patients with VVA. In case of similar vascular architecture, capillary tortuosity and density seem to be comparable. Treatment with topical estrogen results in restoration of the angioarchitecture.