Objectives The upper two-thirds of the vagina is most commonly affected by pelvic organ prolapse. This study aims to find a reference line proximate to the axis of this part of the vagina on MRI which is critical but lacking. Design Retrospective cross-section study Setting Two university-affiliated hospital in China (July 2018 - June 2019) Population Six hundred and fourteen women without prolapse Methods Two reference lines were introduced to compare with the upper two-thirds of the vagina on pelvic MRI: the pubococcygeal line (PCL) and a line passing through the inferior pubic symphysis to the midpoint of the third sacral vertebra (PS3L). Main outcome measures The distances and the angles between the vagina and both reference lines were measured. Results: The median distances from the distal, middle, and apical points to the PS3L were -0.5[interquartile range (IQR), -0.9 - 0.0] cm, 0.0[IQR, -0.4 - 0.6] cm, and -0.2[IQR, -0.9 - 0.0] cm, respectively, while the median distances to the PCL were 0.4[IQR, 0.0 - 0.7] cm, 2.1[IQR,1.7 - 2.5] cm, and 3.1[IQR, 2.5 - 3.7] cm, respectively. The median angle between the vagina and the PS3L was significantly smaller than that with the PCL (0.0[IQR, -4.0 - 7.0] degree vs 29.0[IQR, 23.0 - 34.0] degrees, p<.001). Conclusions: A line passing through the inferior portion of the pubic symphysis to the third sacral vertebra mostly conforms to the normal upper two-thirds of the vaginal axis.