Suppressed parasympathetic nervous system (PSNS) function has been found in a variety of cardiovascular diseases such as hypertension, heart failure and diabetes. However, whether impaired PSNS function plays a significant role in ventricular dysfunction remains to be investigated. Cardiac regulation by the PSNS is primarily mediated by the M₂ muscarinic acetylcholine receptor (M₂-AchR). In this study, we tested the hypothesis that lack of M2-AchR mediated PSNS function may adversely impact cardiac ventricular function. Using M₂-AchR knockout (M₂-AchR KO) and wild type (WT) mice, we found that the basal levels of heart rate and left ventricular function were similar in M₂-AchR KO and WT mice. A bolus injection of Isoproterenol (Iso) induced a greater increase in heart rate in M₂-AchR KO mice than in WT mice. However, the responses of dP/dt to Iso were similar in the two groups. After chronic infusion with Iso for one week, the baseline values of left ventricular function were increased to similar extents in M₂-AchR KO and WT mice. However, the M₂-AchR KO mice exhibited impaired ventricular function, indicated as attenuated dP/dt and increased end diastolic pressure, during an increase in cardiac afterload induced by a bolus injection of phenylephrine (PE). Furthermore, chronic Iso infusion significantly increased matrix metalloproteinase (MMP) activity in the heart in M₂-AchR KO mice. In primary culture of mixed neonatal rat cardiac fibroblast and cardiomyocytes, co-treatment with muscarinic agonist bethanechol (Beth) reversed PE-induced increase in MMP9 activation. These data suggest that M₂-AChR may mediate an inhibitory regulation on MMP function to prevent MMP over activation. The overall results from this study suggest that M₂-AchR mediated PSNS function may provide cardiac protection. Lack of this protective mechanism will increase the susceptibility of the heart to cardiac stresses.