Cow's milk allergy is a common food allergy, with the milk protein αS1-casein being a major allergen. This study aimed to investigate differences in sensitization between cow and goat αS1-CN. Cow and goat αS1-CN were labeled with fluorescent dyes and given to mice sensitized with cholera toxin adjuvant. Both proteins reached immune organs, suggesting no major difference in digestion. However, compared with goat αS1-CN, cow αS1-CN is more readily taken up by dendritic cells, inducing dendritic cell maturation. Furthermore, cow αS1-CN can more effectively induce the generation of Th2 cells, leading to a higher production of specific IgE. In a Caco-2/RBL-2H3 cell model, cow αS1-CN caused more mast cell degranulation and loss of epithelial barrier integrity than goat αS1-CN. In summary, this study found differences in immune responses between cow and goat milk αS1-CN. Cow αS1-CN elicited stronger dendritic cell and Th2 responses, leading to increased mast cell degranulation.