Higher reproductive age is associated with an increased risk of gestational diabetes, pre-eclampsia, and severe vaginal tearing during delivery. Further, menopause is associated with vaginal stiffening. However, the mechanical properties of the vagina during reproductive aging before the onset of menopause are unknown. Therefore, the first objective of this study was to quantify the biaxial mechanical properties of the nulliparous murine vagina with reproductive aging. Menopause is further associated with a decrease in elastic fiber content, which may contribute to vaginal stiffening. Hence, our second objective was to determine the effect of elastic fiber disruption on the biaxial vaginal mechanical properties. To accomplish this, vaginal samples from CD-1 mice aged 2–14 months underwent extension-inflation testing protocols (n = 64 total; n = 16/age group). Then, half of the samples were randomly allocated to undergo elastic fiber fragmentation via elastase digestion (n = 32 total; 8/age group) to evaluate the role of elastic fibers. The material stiffness increased with reproductive age in both the circumferential and axial directions within the control and elastase-treated vaginas. The vagina demonstrated anisotropic mechanical behavior, and anisotropy increased with age. In summary, vaginal remodeling with reproductive age included increased direction-dependent material stiffness, which further increased following elastic fiber disruption. Further work is needed to quantify vaginal remodeling during pregnancy and postpartum with reproductive aging to better understand how age-related vaginal remodeling may contribute to an increased risk of vaginal tearing.