Primary intestinal epithelial cells are a powerful tool to study tight junction protein function and barrier integrity of epithelial cells. However, primary cells are costly, difficult to isolate, have a finite ability to replicate, and require the usage of live tissue. An alternative to primary cells is immortalized cell lines, which are cost effective, easily cultured, stored, and maintain the physiology of primary cells. Several immortalized lines are commercially available, yet there remains a need for cell lines for livestock research, specifically bovine. Here we report an immortalized bovine intestinal epithelial cell (IBEC) line was established by transfection with exogenous human telomerase reverse transcriptase (hTERT). Primary bovine intestinal epithelial cells (PBECs) were isolated from a 2-day old mixed breed calf. PBECs were transfected with hTERT to create an IBEC line. The transfected cells were positive for intestinal epithelial markers while testing negative for fibroblast markers. There was no significant difference in IBEC morphology or physiology post immortalization. In Trans-Epithelial Electrical Resistance (TEER assays), PBECs and IBECs behaved similarly to intestinal porcine enterocyte cells (IPEC-J2) under treatment of Iipopolysaccharide and calcium butyrate. This newly-developed cell line will be an essential and useful tool for in vitro bovine and ruminant intestinal research.