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Development of continuous cell lines has been an indispensable tool for studying disease pathogenesis at cellular and molecular level. Intestinal epithelial cells serve as a barrier between the luminal contents and internal tissue and face unique challenge in maintaining homeostasis. Any form of disruption in the uppermost intestinal epithelium exposes underlying intestinal subepithelial myofibroblast (ISEMF) cells to the luminal content, which results in the generation of immune responses from ISEMF cells. Our main goal was to establish and characterize intestinal epithelial and myofibroblast cell lines from a 3-day old sheep. ISEMF cells play significantly important role in wound healing, tissue regeneration, production of extracellular matrix, epithelial cell migration, and differentiation. There are only few ISEMF cell lines available from ruminant origin. Limited number of studies on innate immune response of ISEMF cells have impeded our understanding of the role of ISMEF cells in innate immunology. In the study, we first established primary ovine ileal epithelial cell (OIEC) cultures and subsequently immortalized primary OIEC with hTERT gene (hTERTOIEC). Both primary and hTERT immortalized OIEC cells displayed typical cobblestone morphology and expressed cytokeratin protein validating their epithelial origin. Epithelial cells were able to polarize, form tight junction proteins and express different surface glycoconjugates. Furthermore, we developed and characterized ovine ileal sub-epithelial myofibroblast cells (ISEMF) from the same 3-day old male lamb and immortalized them using SV40 large T-antigen (SV40-ISEMF). We stimulated primary ovine ISEMF with lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin, imiquimod, poly I:C and poly I:C with lyovec for 3 h and 24 h. We analyzed mRNA expressions of various ovine TLRs (1,2,3,4,5,6 and 9) and cytokines. Upon ligand stimulation, primary ovine ISEMF cells were able to modulate their innate immune responses by expressing variable levels of TLRs and cytokines. Hence, both OIEC and ISEMF cell lines can serve as valuable in vitro tools in elucidating enteric disease pathogenesis.