BackgroundVitamin D (vitD) plays a role in iron metabolism by the suppression of hepcidin, while iron deficiency also impairs vitD metabolism. In undernourished children, iron and vitamin D deficiency are common. There is little knowledge of the inter-relationship between these two nutrients in undernourished and non-undernourished children. AimTo assess the association between 25-hydroxyvitamin D (25(OH)D) and iron status, and the effect of 3 doses of 50,000 IU of vitD on iron status in undernourished and non-undernourished children. MethodsWe measured serum 25(OH)D, haemoglobin (Hb), ferritin and soluble transferrin receptor in 121 undernourished and 51 non-undernourished children in clinics in the North-West Province of South Africa. Three doses of 50,000 IU/week of vitD was supplemented to children with suboptimal vitD levels. ResultsThe overall prevalence of suboptimal vitD concentration (25(OH)D < 30 ng/mL) was 20.3 %. Anaemia and iron deficiency anaemia (IDA) prevalence were 62 % and 49.2 % in the undernourished and 56.9 % and 42.9 % in the non-undernourished group. In the overall group, 25(OH)D < 30 ng/mL was associated with significantly higher IDA prevalence compared to those with 25(OH)D ≥ 30 ng/mL (64.7 % vs 43.0 %, p = 0.023), and was associated with an increased risk of IDA in the crude analysis (OR: 2.434 1.114, 5.318, p = 0.026). Serum 25(OH)D < 30 ng/mL was associated with 4 times (OR: 4.046, 95%CI: 1.022, 16.009, p = 0.046) and greater than 5 times (OR: 5.386, 95%CI: 1.528, 18.985, p = 0.009) increased odds of anaemia and IDA in the undernourished children, respectively. VitD supplementation resulted in a significant reduction of 55.9 % in TNF-α concentration (p = 0.008) in the overall group. ConclusionSuboptimal vitD concentration is associated with increased odds of anaemia and IDA. VitD status should be considered in anaemia prevention strategies, especially in populations where both vitD and iron deficiencies co-exist.