Endometrial tissue is a known source of mesenchymal stem cells (MSCs). We isolated canine endometrial stem cells from canine endometrial tissues using an enzymatic method and confirmed the immunophenotype of mesenchymal stem cells and multilineage differentiation. Canine endometrial tissues were obtained from canine ovariohysterectomy surgery and isolated using 0.2% collagenase type I. We measured the immunophenotype of stem cells using flow cytometry. To confirm the differentiation ability, a trilineage differentiation assay was conducted. In this study, canine endometrialderived MSCs (cEM-MSCs) were isolated by enzyme treatment and showed a spindle-shaped morphology under a microscope. Moreover, cEM-MSCs showed a trilineage differentiation ability. In this study, the canine endometrium was a good source of MSCs.