Congestive heart failure is a very common complication of degenerative heart disease (DVD). Ventricular dysfunction, and ventricular remodelling may also contribute to damage the heart muscle and the progression of the disease appears to be unpredictably. To decelerate the progression and alleviate the symptoms of heart failure using standard treatment seems to be limited, stem cell therapy is a novel option for cardiovascular therapeutic approach. In this study, we examined the therapeutic effects of stem cells from puppy deciduous teeth (pDSCs) in dogs with chronic valvular heart disease. Twenty client-owned dogs with valvular heart disease underwent multiple intravenous injections of allogenic pDSCs. Dogs were assigned to two group: (i) control group with standard treatment, and (ii) group with standard treatment and multiple administrations of pDSCs. Electrocardiography, complete transthoracic echocardiography, thoracic radiography and blood pressure were recorded before and after pDSCs injections for 15, 30 and 60 days. Post pDSCs injection showed measurable improvement in left ventricular ejection fraction, American College of Veterinary Internal Medicine (ACVIM) functional class significantly improved and improved quality of life scores were observed. In the control group, there were no significant enhancements in heart function or ACVIM class. This finding suggests that pDSCs could be an alternative selection for valvular heart disease treatment. However, future studies to understand the underlying mechanism may help to proof the use of pDSCs in veterinary patients with valvular heart diseases.