Pelvic organ prolapse refers to protrusion of the pelvic organ into or out of the vaginal canal. One in four women in the USA suffer from some type of pelvic floor disorder including pelvic organ prolapse and frequency of pelvic organ prolapse is more with increasing age. In Bangladesh, 15.6% women suffered from pelvic organ prolapse and more than 11.0% of women require surgical correction of prolapse in their life times. According to few researches, there is an observed association between low vitamin D levels with pelvic organ prolapse but this finding is not unequivocal. This case control study has been conducted in the Department of Obstetrics and Gynaecology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Bangladesh from October 2018 to August 2020 to investigate the level of vitamin D in patients with and without pelvic organ prolapse to explore the association of low vitamin D with pelvic organ prolapse. A total 148 participants of 52 years or older attending the out or inpatient department were enrolled in the study. The study population was divided into two groups, a case group (n=74) consisting of patients with pelvic organ prolapse and a control group (n=74) comprising of women without pelvic organ prolapse. After taking informed written consent the serum vitamin D level of all participants was measured by CMIA technology with flexible assay protocols at Biochemistry and Molecular Biology department of the same institute. All necessary Data were collected on variables of interest by using the structured questionnaire pre-designed for interview, observation, clinical examination, and biochemical Data collection. Distributions were expressed by mean and standard deviation for continuous variables and by frequency and percentage for qualitative variables. Student's t-test and Chi square test were done to see the significance of differences between Group I and Group II. Odds ratio, correlation coefficient, and multivariate logistic regression analysis was done to assess the association of low vitamin D level with pelvic organ prolapse. Mean±SD level of Vitamin D in the case group was 13.96±5.18ng/ml and in the control group was 21.08±5.77ng/ml respectively. The difference was statistically significant (p<0.05). Moreover, the vitamin D levels were inversely proportionate with the severity of pelvic organ prolapse. OR (95% CI), of two groups showed risk of developing pelvic organ prolapse 5.63 times higher in women with decreased vitamin D level. Thus it can be concluded that women having low level of vitamin D have more chance of developing pelvic organ prolapse.