Background: Pregnant women are at a high risk of iron deficiency anaemia in Ghana. Despite the provision of iron nutrition education and supplementation for the prevention of iron deficiency anaemia in pregnancy, the estimated prevalence of iron deficiency anaemia in pregnancy is relatively high. The associated factors such as maternal knowledge of iron nutrition and supplementation therapy are not clear due to limited data. Objective: This study aims to assess maternal knowledge of iron nutrition and supplementation and associated factors in the rural Tain District of Ghana. Method: Cross-sectional quantitative data was used. Data was collected from a sample of 480 pregnant women attending antenatal clinics in any of the health facilities in the Tain district. The ten health facilities were stratified and the allocation of samples to each of the ten facilities was proportionate to size. Simple random sampling was used to select individual participants from a compiled list of registered antenatal mothers in each health facility. Results: In all, 36% of participants reported having adequate knowledge of iron nutrition and supplementation. Pregnant women whose husbands have no formal/Primary education (AOR=0.0078(95% CI: 0.021, 0.294), Middle/JHS education (AOR=0.223(95% CI: 0.067, 0.743) were associated with a knowledge of iron nutrition and supplementation. Pregnant women who have one child (AOR= 0.150 (95% CI: 0.053, 0.426) and those who have two children (AOR= 0.375(95% CI: 0.146, 0.963) had lower odds of adequate knowledge of iron nutrition and supplementation. Conclusion: Maternal knowledge of iron nutrition and supplementation for the prevention of iron deficiency anaemia is low. Health education on iron nutrition and supplementation therapy should be strengthened.