Deoxynivalenol (DON) is one of the most widely distributed mycotoxins in the food chain, and the protective effect of kaempferol (KAM) pretreatment against the barrier dysfunction induced by DON in a Caco-2 cell model was investigated using a proteomic approach. The results showed that after KAM pretreatment, both the numbers of down- and up-regulated differentially expressed proteins were significantly lower than those in the DON group. Gene ontology analysis revealed that differentially expressed proteins were enriched in cell adhesion molecule binding, cell junction, and cell junction assembly. Further study demonstrated that KAM pretreatment affected the expression and assembly of tight junction and adherens junction proteins through the PKA pathway and MAPK/ERK pathway to improve the barrier integrity of the Caco-2 cell monolayer, and nutraceutical approach based on bioactive phytochemicals to improve the body's immunity might be an effective strategy to combat the adverse effects of mycotoxins on intestinal barrier function.