Background and objectives: Female reproductive function flexibly responds to ecological variation in energy availability, but the roles of other ecologically limited resources, such as iron, remain poorly understood. This analysis investigates whether haemoglobin associates with investment in reproductive function in a rural natural fertility population living in the Bolivian altiplano. Methodology: We conducted a cross-sectional secondary analysis of prospectively collected biomarker and sociodemographic data, comprising 152 menstrual cycles from 96 non-contracepting women living at 3800 m altitude. Multivariable multilevel models were used to investigate (i) whether haemoglobin concentration is associated with ecological variation in subsistence strategy and seasonal conditions, and (ii) whether haemoglobin concentration is associated with the occurrence of ovulation and/or the concentration of luteal progesterone, two biomarkers of current investment in reproduction. Results: Haemoglobin concentrations were lower in arduous seasons among those women more dependent on traditional agropastoral subsistence strategies (β = −0.42, 95% CI: −0.80 to −0.04, P = 0.032). During more arduous seasons, a 1 standard deviation increase in haemoglobin was associated with an over 3-fold increase in the odds of ovulation after adjusting for body fat, breastfeeding status, and age (adjusted odds ratio = 3.27, 95% CI: 1.10 to 9.27, P = 0.033). Conclusions and implications When conditions are relatively harsh and may be expected to improve, low haemoglobin levels are associated with lower current investment in reproduction and reduced fecundity. These results support the role of iron, independent of energy stores, as a limiting resource in modulating reproductive trade-offs.