BACKGROUND: The role of genetic determinants in mediating iron status in Africans is not fully understood. Genome-wide association studies in non-African populations have revealed genetic variants in the transmembrane protease serine 6 gene (TMPRSS6) that are associated with the risk of anemia. OBJECTIVES: To investigate the effects of risk alleles for low iron status, namely TMPRSS6 rs2235321, rs855791, and rs4820268, on responses to oral iron in healthy Gambian adults. METHODS: Using a recall-by-genotype design, participants were selected from a pregenotype cohort of 3000 individuals in the Keneba Biobank (Medical Research Council Unit The Gambia at the London School of Hygiene & Tropical Medicine). Participants were invited to participate in the study based on 9 genotype combinations obtained from 3 TMPRSS6 single nucleotide polymorphisms (SNPs): rs2235321, rs855791, and rs4820268. The participants fasted overnight and then ingested a single oral dose of ferrous sulfate (130 mg elemental iron). Blood samples were collected prior to iron ingestion and at 2 and 5 h after the oral iron dose. The effects of genotype on hepcidin and plasma iron parameters were assessed. RESULTS: A total of 251 individuals were enrolled. Homozygous carriers of the major TMPRSS6 alleles at each of the SNPs had higher plasma hepcidin at baseline (rs2235321: GG compared with AA = 9.50 compared with 6.60 ng/ml, P = 0.035; rs855791: GG compared with AG = 9.50 compared with 4.96 ng/mL, P = 0.015; rs4820268: AA compared with GG = 9.50 compared with 3.27 ng/mL,  P = 0.002) and at subsequent timepoints. In most subjects, hepcidin concentrations increased following iron ingestion (overall group mean = 4.98 ± 0.98 ng/mL at 5 h, P < 0.001), but double heterozygotes at rs2235321 and rs855791 showed no increase (0.36 ± 0.40 ng/mL at 5 h, P = 0.667). CONCLUSIONS: This study revealed that common TMPRSS6 variants influence hepcidin concentrations, but not iron status indicators either at baseline or following a large oral dose of iron. These results suggest that genetic variations in the TMPRSS6 gene are unlikely to be important contributors to variations in iron status in Africans.This study was registered at clinicaltrials.gov (# NCT03341338).