The effect of the horn status of cows on their milk composition and quality is a controversial research topic. In this study, 128 milk samples from 64 horned and 64 disbudded Brown Swiss and Original Braunvieh cows were collected from alpine farms where both horned and disbudded cows were grazing on mountain pastures. The samples were analyzed for their detailed composition and protein digestion in a simulated in vitro digestion (INFOGEST). To exclude probable influences on digestion, the β-casein genotype with its variants A1 and A2 was also included in the study. The effects of horn status and β-casein genotype were investigated in linear mixed models, which included additional influencing random factors such as Original Braunvieh blood proportion, stage of lactation, and farm. Horn status did not have any effect on milk composition or digestion. In contrast, milk from A1A1 cows showed a different protein digestion than milk of A1A2 and A2A2 cows in the gastric phase, including smaller amounts of β-casomorphin(BCM)21-associated peptides and larger amounts of BCM11-associated peptides. Abundances of BCM7 did not differ between β-casein genotypes. At the end of the intestinal phase, the digested milk of A1A1 and A2A2 b-casein genotypes did not differ.