Multivitamin corn is a novel genetically engineered variety that simultaneously produces high levels of β-carotene, ascorbate and folate, and therefore has the potential to address simultaneously multiple micronutrient deficiencies caused by the lack of vitamins A, B9 and C in developing country populations. As part of the development process for genetically engineered crops and following European Food Safety Authority (EFSA) recommendations, multivitamin corn must be tested in whole food/feed sub-chronic animal feeding studies to ensure there are no adverse effects, and potential allergens must be identified. We carried out a 28-day toxicity assessment in mice, which showed no short-term sub-acute evidence of diet-related adverse health effects and no difference in clinical markers (food consumption, body weight, organ/tissue weight, haematological and biochemical blood parameters and histopathology) compared to mice fed on a control diet. A subsequent 90-day sub-chronic feeding study again showed no indications of toxicity compared to mice fed on control diets. Our data confirm that diets enriched with multivitamin corn have no adverse effects on mice, do not induce any clinical signs of toxicity and do not contain known allergens.