The bovine gastrointestinal (GI) epithelium plays a central role in nutrient absorption, barrier regulation, and host–microbe interactions; however, primary cell-based in vitro systems for bovine GI tissues remain limited. Isolation and maintenance of primary gastrointestinal epithelial cells are challenging due to tissue complexity, microbial contamination risk, and culture-associated phenotypic changes. This study aimed to isolate, culture, and perform a preliminary marker-based characterization of epithelial-enriched primary bovine gastrointestinal cell cultures derived from four GI tract segments: rumen, jejunum, ileum, and colon. Gastrointestinal tissues were collected from three 4-month-old Holstein calves. Luminal mucosal tissue was mechanically separated, enzymatically dissociated, and cultured in vitro. Initial culture performance outcomes, including post-cell isolation viability, were recorded. Epithelial cell-associated gene expression was evaluated using qPCR for CDH1, CLDN4, TJP1, KRT8, and KRT18 at three stages: whole tissue, post-cell isolation, and post-cell culture. Keratin 8 (KRT8) staining was assessed by immunofluorescence in pooled cultured cells, with descriptive quantification of KRT8-positive cells in the available images. Linear mixed-effects analysis accounting for repeated measurements within calf identified an effect of experimental stage for all five transcripts after false-discovery-rate correction. Overall effects of Segment and the Segment × Stage interaction were retained only for TJP1, indicating that stage-associated expression patterns were not uniform among gastrointestinal regions for this marker. KRT8 immunofluorescence demonstrated KRT8-positive cells in cultures derived from all four gastrointestinal segments. Across eight quantified fields, 244 out of 325 DAPI-defined cells (75.1%) were classified as KRT8-positive. These findings support the isolation and culture of epithelial-enriched primary bovine GI cultures under the conditions tested. While additional standardized culture-performance assessment, expanded characterization of epithelial and non-epithelial cell populations, passage stability analysis, and functional barrier assays are needed, this work provides a preliminary methodological framework for epithelial-enriched primary bovine GI cell cultures and supports their future application in studies of epithelial biology, host–microbe interactions, and molecular responses of the bovine gastrointestinal tract.