Objectives Isolation and culture of distinct primary endometrial cells are key to reliable in-vitro models to investigate the uterine immune response and optimse new disease interventions. Details on the isolation method and purity of distinct cell populations is lacking in currently available protocols leading to inconsistent results across laboratories. Methods Bovine endometrial tissue from non-pregnant bovine uteri were collected immediately post-mortem and separated using differential size filtering. Isolations ( n = 15) yielded an average of 3.1 × 105 ± 0.7 × 105 epithelial cells and 1.88 × 106 ± 5.44 × 105 stromal fibroblasts per uterine horn. Following expansion in culture, the purity of cell populations was confirmed using morphology and positive staining for cytokeratin and vimentin which identifies epithelial and stromal fibroblast populations, respectively. Using PCR, cDNA from both cell populations was negative for CD45, a marker of immune cells. Results On challenge with a bacterial PAMP (LPS), epithelial and stromal fibroblasts showed a marked increase in the expression of the inflammatory mediators IL8 , IL6 , S100A8 and S100A9, with both cell populations displaying distinct expression profiles. Here we provide a detailed methodology on the culture of primary bovine endometrial epithelial and stromal cells and demonstrate these cells provide a physiologically relevant model for studies of endometrial inflammation and its regulation. Electronic supplementary material The online version of this article (10.1007/s11259-020-09770-3) contains supplementary material, which is available to authorized users.