Background/Aims: To compare biomechanical properties of vaginal tissues between women with and without pelvic organ prolapse (POP) and investigate factors that may influence these properties. Methods: Forty patients submitted to POP surgery and 15 non-POP cadavers were evaluated. The tissue was excised from anterior and posterior middle third vagina. The biomechanical properties considered were stiffness (E) and maximum stress (Smax), and they were evaluated by means of uniaxial tension tests. Results: POP patients were associated with higher values of E (13.1 ± 0.8 vs. 9.5 ± 0.7 MPa; p < 0.001) and Smax (5.3 ± 0.5 vs. 3.2 ± 0.9 MPa; p < 0.001) in the anterior vaginal wall compared to the posterior wall. In contrast, non-POP women presented lower values of E (6.9 ± 1.1 vs. 10.5 ± 1.0 MPa; p = 0.01) and Smax (2.6 ± 0.4 vs. 3.5 ± 0.4 MPa; p = 0.043) in the anterior wall. The occurrence of POP was the only independent predictor of higher values of E and Smax in anterior vaginal samples (p = 0.003 and p = 0.008, respectively). Women with severe anterior vaginal prolapse presented higher levels of E and Smax in the anterior sample compared to those with lower POP stages (p = 0.001 and p = 0.01; respectively). Conclusion: Women with POP present significant changes of biomechanical properties in the vagina.