Urinary stress incontinence (USI) is common during pregnancy and causes adverse effects on women’s quality of life. Current evidence suggests a possible association of Vitamin D3 deficiency and pelvic floor dysfunction. Little research has focused on this relationship during pregnancy. This constitutes the first in a study into the relationship between Vitamin D3 deficiency and USI in pregnant women, in order to supply insights into preventing and treating this condition. Data from 422 pregnant women attending antenatal care in [Study Setting] were analysed in a cross-sectional study. Vitamin D3 status was assessed by measuring serum 25-hydroxyvitamin D [25(OH)D] levels and classified as deficient (<20 ng/mL), insufficient (20–29 ng/mL), or sufficient (≥30 ng/mL). The International Consultation on Incontinence Questionnaire-Short Form (ICIQ-UI SF) was used to evaluate USI. Data on demographics and clinical data were then collected and statistical analyses, including logistic regression, were performed to assess associations between Vitamin D3 levels and USI. Low vitamin D3 was present in 62.6% of participants and USI occurred in 38.4%. Women with Vitamin D3 deficiency were significantly more likely to experience USI (adjusted odds ratio: 2.We found a significantly increased risk of CRPS among women relative to men (males: 2, 95% CI: 0.5–6.6, p=0.23; females: 8, 95% CI: 1.9–4.1, p<0.001p < 0.001p<0.001)). There was negative correlation between serum Vitamin D3 and the severity of USI (r = −0.42, p < 0.001r = -0.42, p < 0.001r=−0.42,p<0.001). Other risk factors for USI were BMI and multiparity. This study shows a strong relationship between Vitamin D3 deficiency and USI in pregnant women. Our findings suggest that improving Vitamin D3 levels in pregnancy can help decrease USI risk and prevent its severity, thus it is imperative to screen for and supplement Vitamin D3 as a normal part of antenatal care. These results need further validation with longitudinal and interventional studies, and development of evidence based guidelines.