Exome sequencing has identified the glyceronephosphate O-acyltransferase (GNPAT) gene as a genetic modifier of iron overload in hereditary hemochromatosis. Subjects with HFE (Homeostatic Iron Regulator) p.C282Y mutations and the GNPAT p.D519G variant had more iron loading compared to subjects without the GNPAT variant.  In response to an oral iron challenge, women with GNPAT polymorphisms loaded more iron as compared to women without polymorphisms, reinforcing a role for GNPAT in iron homeostasis. The aim of this study was to develop and characterize an animal model of disease to further our understanding of genetic modifiers, and in particular, the role of GNPAT in iron homeostasis. We generated a Hfe/Gnpat mouse model reminiscent of the patients previously studied and studied these mice for up to 26 weeks. We also examined the effect of dietary iron loading on mice with reduced Gnpat expression. Gnpat heterozygosity in Hfe knockout mice does not play a role in systemic iron homeostasis; Gnpat+/- mice fed a high-iron diet, however, had lower hepatic hepcidin mRNA expression, whereas they have significantly higher serum iron levels and transferrin saturation compared to wildtype littermates on a similar diet. These results reinforce an independent role of GNPAT in systemic iron homeostasis, reproducing in an animal model the observations in women with GNPAT polymorphisms subjected to an iron tolerance test.