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Objective To determine whether 25OH-D levels in the follicular fluid (FF) of infertile women undergoing IVF demonstrate a relationship with IVF cycle parameters and outcome, hypothesizing that levels of 25OH-D in body fluids are reflective of vitamin repletion status. Design Prospective cohort study. Setting Academic tertiary care center. Patient(s) Eighty-four infertile women undergoing IVF. Intervention(s) Follicular fluid from follicles ≥14mm; serum (n = 10) and FF levels of 25OH-D. Main Outcome Measure(s) Clinical pregnancy (CP), defined as evidence of intrauterine gestation sac on ultrasound, following IVF; IVF cycle parameters. Result(s) Serum and FF levels of 25OH-D were highly correlated (r = 0.94). In a predominantly Caucasian population (66%), significantly lower FF 25OH-D levels were noted in Black versus non-Black patients. Significant inverse correlations were seen between FF 25OH-D levels and body mass index (r = –0.25). Significantly higher CP and implantation rates were observed across tertiles of FF25OH-D; patients achieving CP following IVF (n = 26) exhibited significantly higher FF levels of 25OH-D. Multivariable logistic regression analysis confirmed FF 25OH-D levels as an independent predictor to success of an IVF cycle; adjusting for age, body mass index, ethnicity, and number of embryos transferred, each ng/mL increase in FF 25OH-D increased the likelihood for achieving CP by 6%. Conclusion(s) Our findings that women with higher vitamin D level in their serum and FF are significantly more likely to achieve CP following IVF–embryo transfer are novel. A potential for benefit of vitamin D supplementation on treatment success in infertile patients undergoing IVF is suggested and merits further investigation.