Abstract Study question Is there an association between iron intake and ovarian reserve among women seeking fertility care? Summary answer Supplemental iron intake above 45mg/d is associated with lower ovarian reserve among women seeking fertility care. What is known already Although the literature regarding iron intake in relation to ovarian reserve is scant and inconsistent, some evidence suggest that iron may have gonadotoxic effects. Study design, size, duration Observational study including 582 female participants attending the Massachusetts General Hospital Fertility Center (2007–2019) enrolled in the Environment and Reproductive Health (EARTH) Study Participants/materials, setting, methods Iron intake was estimated using a validated food frequency questionnaire (FFQ). Markers of ovarian reserve included antral follicle count (AFC) (assessed via transvaginal ultrasound) and day 3 FSH, both obtained during the course of infertility evaluation. The association between iron intake and ovarian reserve was evaluated using Poisson regression models for AFC and quantile regression models for day 3 FSH adjusted for age, menstrual cycle characteristics, physical activity, carbohydrate intake and total energy intake. Main results and the role of chance Participants had a median age of 35 years and median total iron intake of 29 mg/d. Total iron intake was inversely related to AFC and this association was driven by intake of supplemental iron. Compared to women with a supplemental iron intake ≤20mg/day, women consuming 45-64mg/d of supplemental iron had a 17% (-35%, 0.3%) lower AFC and women consuming ≥65mg/d of supplemental iron had a 32% (-54%, -11%) lower AFC after adjusting for potential confounders (p, linear trend=0.003). Similarly, in multivariable-adjusted analysis, day 3 FSH levels were 0.9 (0.5, 1.3) IU/mL higher among women with a supplemental iron intake ≥65mg/d when compared to women with a supplemental iron intake ≤20mg/d (p, linear trend=0.02). Limitations, reasons for caution Iron intake was estimated using a method that relies on self-report and we had no biomarkers of iron status in our participants. Also, only 36 women consumed ≥45mg/d of supplemental iron. Wider implications of the findings Since all study participants were seeking fertility treatment, our findings may not apply to women in the general population. Although our findings are consistent with studies of women with iron overload, given the paucity of literature on this topic it is essential that this question is revisited in future studies. Trial registration number Not applicable, not a randomized trial