Enterotoxigenic Escherichia coli (ETEC) F4 causes diarrhea in infants and weaned piglets. The technique of isobaric tags for relative and absolute quantitation (iTRAQ) was used in this study to determine the differentially expressed proteins in porcine intestinal epithelial cells (IPEC-J2) after pretreatment with Lactobacillus plantarum (LP) followed by challenge with ETEC F4. A total of 4771 proteins were identified in IPEC-J2 cells, with 90, 105, and 134 differentially expressed proteins in cells exposed to ETEC, LP, and LP + ETEC, respectively. The COG analysis divided the identified proteins into 20 categories. The GO and KEGG annotation indicated that most of the differentially expressed proteins were enriched in various biological metabolism including cell cycle control, cell division and differentiation. Additionally, western blotting analyses confirmed the reduced abundance of selected proteins of the mTOR and MAPK signal pathways affected by ETEC F4. Moreover, LP pretreatment increased JNK activation in IPEC-J2 cells infected with ETEC F4. These results may provide further insights into the mechanisms involved in the interaction between ETEC F4 and intestinal epithelial cells, and broaden the understanding of the protective effects of LP in alleviating ETEC-provoked diarrhea of piglets.Copyright © 2019. Published by Elsevier B.V.