This research comprehensively examines 88 infant formulas available in the US market, with an emphasis on their diverging nutritional attributes based on lactose content. We stratified formulas into three categories: lactose-free, lactose-reduced, and entirely lactose-based. The formulas’ nutritional content for 58 nutrients was obtained from the Nutrition Data System for Research (NDSR). Nutritional analysis revealed significant differences in nutrient composition across formula categories. For example, the results showed significant associations between the lactose content and glycemic index (GI) of the formula as well as 25 other nutrients. Specifically, we showed that for every gram of lactose per 100 g of formula that is removed, there was a 10.1% increase in GI (β = −10.12, p ≤ 0.000), a 19%,5%, and a 2% increase in added sugar (β = −0.19, p < 0.01), protein (β = −0.05, p < 0.001), and polyunsaturated fatty acids (β = −0.01, p < 0.01). The substitution of lactose in infant formulas significantly alters their nutritional profile, inducing changes in GI, added sugar, protein, and polyunsaturated fatty acids. These modifications have potential consequences for infant growth and metabolic responses and could influence long-term health trajectories. The clinical relevance of the composition differences between formulas should be further explored.