Epidermal growth factor (EGF) and glucagon-like peptides (GLP) modulate the tight junctions (TJ) of the intestinal epithelial barrier (EB) of monogastric animals. This work tried to elucidate whether GLP-1, GLP-2 and EGF can affect the EB of the rumen. Ovine ruminal epithelia were incubated in Ussing chambers for 7 hr with 25 or 250 nM of either GLP-1 or GLP-2 on the serosal side, with 2.5 nM of EGF on the serosal side or with 0.25 or 2.5 nM EGF on the mucosal side. No treatment affected tissue conductance. Short-circuit current (Isc ) was affected by time and treatment and their interactions. Only 250 nM of either GLP-1 or GLP-2 decreased Isc in certain periods compared with 25 nM GLP-1 or 0.25 nM mucosally applied EGF; however, not when compared to control epithelia. Fluorescein flux rates (Jfluor ) of ruminal epithelia were affected by treatment, time and time × treatment interaction. The time × treatment interaction was based on an increase in Jfluor between the first and last hour in epithelia incubated with 25 nM GLP-1 or GLP-2 and in epithelia incubated with EGF. After 7 hr incubation, claudin-7 mRNA expression was downregulated in all treatments. Claudin-1 mRNA was upregulated after incubation with 2.5 nM EGF on the serosal side, claudin-4 mRNA was downregulated by 2.5 nM EGF on the mucosal side, and occludin mRNA was increased after incubation with 250 nM GLP-2. The protein abundance of all tested TJ proteins was not influenced by treatment. We conclude that GLP-1, GLP-2, and EGF have no obvious acute effects on the EB of ruminal epithelia under simulated physiological conditions ex vivo. However, by decreasing the mRNA expression of claudin-7 and partly affecting other TJ proteins, they may modulate EB in the longer term or under certain conditions.,© 2019 The Authors. Journal of Animal Physiology and Animal Nutrition published by Blackwell Verlag GmbH.