Vitamin D affects skeletal muscle strength and functions via various mechanisms. Strength and/or functional dysfunctions of the pelvic floor muscles may be associated with the distortion of pelvic floor functions. We hypothesized that vitamin D deficiency may contribute to pelvic floor dysfunction (PFD) by affecting pelvic floor muscle strength (PFMS). The aim of this study was to assess the effect of vitamin D deficiency during pregnancy on postpartum PFMS. This cross-sectional study was conducted in a university hospital. One hundred and eighty pregnant women were admitted to our hospital in their third trimester and compared with 156 healthy nulliparous women. Venous blood samples for examining vitamin D levels were taken from each participant and stored at -80 °C. At 8-10 weeks postpartum, patients were invited to the hospital, asked about their PFD symptoms, and PFMS was measured using a perineometer. There was no statistical significance among groups regarding mean age, maternal age, and weight at delivery. Postpartum PFMS and duration in vitamin D-deficient women were significantly lower than those without the deficiency. Vitamin D-deficient vaginal delivery cases (group I) had a postpartum PFMS average of 21.96 ± 7.91 cm-H₂O, nonvitamin D-deficient normal delivery cases (group III) had a PFMS of 29.66 ± 10.3 cm-H₂O (p = 0.001). In the cesarean delivery groups, vitamin D-deficient (group II) and nonvitamin D-deficient (group IV) cases had PFMS values of 32.23 ± 9.66 and 35.53 ± 15.58 cm-H₂O respectively (p = 0.258). Lower vitamin D levels in the third trimester correlates with decreased PFMS.