Children who grow up on farms have a lower risk of developing childhood atopic disease. Although human milk proteins are heavily glycosylated, there is a lack of studies investigating the milk glycoproteome. In this study, we have used label-free quantitative proteomics to analyze milk samples from Rochester and Older Order Mennonite mothers, two populations with different lifestyles, exposures, and risk of allergic disease. We identified multiple N-glycopeptides with significantly different abundances between the two communities and four N-glycopeptides that may have a protective effect against the development of atopic disease. The findings of this study indicate that the differential glycosylation of milk proteins may affect the development of atopic disease, something previously uninvestigated.