Here, we provide a protocol for reliable isolation and subculture of putative mesenchymal stem cells from mice colons. This method provides a good approach to cultivate and characterize putative colonic mesenchymal stem cells (cMSCs). A high purity of cMSCs can be obtained according to this protocol. The whole isolation processes of cMSCs take about 2 h with two important digestion steps involved. Only with common culture medium, maturation of cMSCs in culture proceeds approximately 2 weeks to allow relevant researches to be conducted. This protocol sheds light on better cultivation of MSCs in vitro from post-natal colon tissues. These putative cMSCs share common phenotypic property with those in vivo reported, and contain potential lineage differentiation capacity. The successful culture of cMSCs in vitro provides an ideal model for study of MSCs biology in the intestine.