In this study, we determined whether multilineage‐differentiating stress‐enduring (Muse) cells exist in rat bone marrow and elucidated their effects on protection against the injury of intestinal epithelial cells associated with inflammation. Rat Muse cells were separated from bone marrow mesenchymal stem cells (BMMSCs) by trypsin‐incubation stress. The group of cells maintained the characteristics of BMMSCs; however, there were high positive expression levels of stage‐specific embryonic antigen‐3 (SSEA‐3; 75.6 ± 2.8%) and stage‐specific embryonic antigen‐1 (SSEA‐1; 74.8 ± 3.1%), as well as specific antigens including Nanog, POU class 5 homeobox 1 (OCT 3/4), and SRY‐box 2 (SOX 2). After inducing differentiation, α‐fetoprotein (endodermal), α‐smooth muscle actin and neurofilament medium polypeptide (ectodermal) were positive in Muse cells. Injuries of intestinal epithelial crypt cell‐6 (IEC‐6) and colorectal adenocarcinoma 2 (Caco‐2) cells as models were induced by tumor necrosis factor‐α stimulation in vitro. Muse cells exhibited significant protective effects on the proliferation and intestinal barrier structure, the underlying mechanisms of which were related to reduced levels of interleukin‐6 (IL‐6) and interferon‐γ (IFN‐γ), and the restoration of transforming growth factor‐β (TGF‐β) and IL‐10 in the inflammation microenvironment. In summary, there were minimal levels of pluripotent stem cells in rat bone marrow, which exhibit similar properties to human Muse cells. Rat Muse cells could provide protection against damage to intestinal epithelial cells depending on their anti‐inflammatory and immune regulatory functionality. Their functional impact was more obvious than that of BMMSCs.