During the thermal processing of milk, Maillard reactions occur between proteins and lactose to generate glycated proteins. In this study, a lactose-glycated caseinate was hydrolyzed by trypsin. The obtained glycated caseinate (GCN) hydrolysate had a lactose content of 10.8 g/kg of protein. We identified its glycation sites and then assessed it for its protective effect against lipopolysaccharide-induced barrier injury using a rat intestinal epithelial cell line (IEC-6 cells) as a cell model and unglycated caseinate (CN) hydrolysate as a reference. Results from our liquid chromatography–mass spectrometry analysis of the GCN hydrolysate verified that lactose glycation occurred at the Lys residues in 3 casein components (α_S1-casein, β-casein, and κ-casein), and this resulted in the formation of 5 peptides with the following amino acid sequences: EMPFPKYPKYPVEPF, HIQKEDVPSE, GSENSEKTTMPL, NQDKTEIPT, and EGIHAQQKEPM. The results from cell experiments showed that the 2 hydrolysates could promote cell growth and decrease lactate dehydrogenase release in the lipopolysaccharide-injured cells; more importantly, they could partially protect the damaged barrier function of the cells by increasing trans-epithelial electrical resistance, decreasing epithelial permeability, and upregulating the expression of the 3 tight junction proteins zonula occludens-1, occludin, and claudin-1. However, compared with CN hydrolysate, GCN hydrolysate showed lower efficacy in protecting against cellular barrier dysfunction. We propose that the different chemical characteristics of the CN hydrolysate and the GCN hydrolysate (i.e., amino acid loss and lactose conjugation) contributed to the lower barrier-protective efficacy of the GCN hydrolysate. During dairy processing, protein glycation of the Maillard type might have a non-negligible, unfavorable effect on dairy proteins, in view of the resulting protein glycation we found and the critical function of proteins for maintaining the integrity of the intestinal barrier.