Objective Comparison of the levator ani muscles in three-dimensional (3D) MRI-based models in women with and without pelvic organ prolapse at rest to analyze the morphological characteristics of levator ani muscles in women with POP. Methods Twenty-five women with POP and 22 women with normal pelvic support were selected from Nanfang Hospital of Southern Medical University. Axial, sagittal, and coronal T2-weighted pelvic magnetic resonance scans were obtained with the women in the supine position.The 3D models were reconstructed from the source images. Morphological changes was compared within the two groups of levator ani muscles, and the 3D models were measured to determine the levator ani muscle volume (LVOL), levator plate angle (LPA), levator hiatus width (LH-W) and length (LH-L), distance between symphysis and levator sling muscle (LSG). Results There were no puborectalis avulsions in control, in POP, 3 cases of avulsions just in left, 3 cases of avulsions just in right, 7 cases in bilateral. The shape of iliococcygeus were all dome-shaped in control, 11 cases were U-shaped and 14 cases were dome-shaped in POP. The shape of levator hiatus were 7 cases of U-shape, 12 cases of V-shape, 3 cases of irregular in control; 5 cases of U-shape, 4 cases of V-shape, 16 cases of irregular in POP. POP versus control: LH-L: (68.0 ± 8.9) versus (61.6 ± 7.2) mm (P<0.05); LH-W: (41.4 ± 3.9) versus (38.0 ± 3.2) mm (P<0.05); LSG-L: (29.6 ± 7.4) versus (24.6 ± 3.7) mm (P<0.05); LSG-R: (28.4 ± 6.8) versus (23.9 ± 3.2) mm (P<0.05); LPA: (51.0 ± 11.3)° versus (40.6 ± 6.3)° (P<0.05); LVOL: (23.7 ± 5.8) versus (24.6 ± 5.0) cm3 (P>0.05). Conclusions It is possible to assess the morphologic changes of levator ani by using 3D MRI models objectively, our 3D data demonstrate larger in LVOL, LPA, LH-W, LH-L, LSG, and the changes in shape. It is helpful to diagnose and assess the specific situation of patients POP in clinic.