Intestinal subepithelial myofibroblasts (ISMFs) are mesenchymal cells that exist under the epithelium of intestines. Primarily isolated ISMFs from rodents have been applied to experiments. However, due to the size of their intestines, the available cell number is limited. Thus, we attempted to isolate ISMFs from bovine colon as an alternative material. After detachment of smooth muscle and epithelial layers, colonic mucosa was explanted. After 2-week incubation, α-SMA (+) / vimentin (+) / desmin (−) ISMFs were harvested and applied for experiments. First we examined the effect of cell passage on morphology and proliferation activity of bovine ISMFs. Although 3rd and 7th passage bovine ISMFs did not exhibit any changes, 11th passage ISMFs showed rounded enlarged shape and lost proliferation potential. On the contrary, rat ISMFs displayed the above senescent changes at earlier passage (passage 4). In intracellular Ca²⁺ concentration measurement, bioactive substances (0.3 – 1 µM ATP, 0.1 – 1 µM serotonin, 10 – 100 nM endothelin-1, and 1 – 10 nM bradykinin) dose-dependently induced an increase in intracellular Ca²⁺ concentration in bovine ISMFs (passage 3 and 7). However, at passage 11, impairment in intracellular Ca²⁺ responses was observed. Thus, bovine ISMFs might be a novel useful tool with long life span and good cellular responses to investigate physiological/pathophysiological roles of ISMFs.