Providing adequate vitamin D3 for the human diet is a long-standing goal of crop breeding because most plants have relatively low levels of this nutrient naturally. Using CRISPR-Cas9 editing of the tomato genome, we increased provitamin D3 levels in tomato fruits to up to 1 mg per 100 grams dry weight. Use of this provitamin D3 tomato could ameliorate widespread vitamin D deficiencies around the world.