The rumen and its function gain more and more importance in cattle industry. Only a limited number of studies are available, using primary rumen epithelial cells isolated from cows (Sun et al., 2017; Stumpf et al., 2009). Establishing a cell based rumen in vitro model provides an important tool to investigate not only the rumen metabolism, but will also help to evaluate the effects of different toxins (e.g., bacterial toxins, mycotoxins) on rumen cells. The aim of the study was to isolate, cultivate, and characterize rumen epithelial cells (REC) isolated from the rumen tissue of dairy cows. Furthermore, it was assessed, if deoxynivalenol (DON) and fumonisin B₁ (FB₁) affect cell viability of REC. Rumen tissue was obtained from a local abattoir and transported on ice to the laboratory. Epithelial cells were isolated from the tissue by enzymatic dissociation with trypsin. Cell growth was visually checked with a light microscope. In addition, cells were characterized via immunostaining for cytokeratin and tight junction proteins. The water-soluble tetrazolium salt (WST-1) assay was used to assess the effects of 2 mycotoxins on cell viability: DON (0–25 µM) and FB₁ (0–25 µM). For statistical evaluation of data, GraphPad Prism software (Version 7) was used. As data were normally distributed, ANOVA was performed with Dunnett’s as post-hoc test. P-values of <0.05 were considered as significant. REC were successfully isolated from the rumen tissue and could be cultivated for up to 8 passages without changes in cell morphology. Cells were positively stained for cytokeratin (epithelial cell marker) and all 3 tested tight junction proteins (claudin-1, occludin and zonula occludens-1). DON significantly decreased cell viability at a concentration of 1 µM (P < 0.05), whereas FB₁ significantly decreased cell viability at 12.5 µM (P < 0.05).The establishment of an in vitro model with REC was successful as cells could be cultivated and showed cell type specific characteristics. Furthermore, DON seems to exhibit higher cytotoxicity than FB₁ in REC.