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 (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 change in pressure over time (dP/dt) to Iso were similar in the two groups. After chronic infusion with Iso for 1 wk, 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. 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, cotreatment with muscarinic agonist bethanechol reversed phenylephrine-induced increase in MMP-9 activation. These data suggest that M₂-AChR may mediate an inhibitory regulation on MMP function. 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.