Previous studies showed that circulating autoantibodies against M muscarinic receptors (anti-M R Ab) are associated with decreased cardiac parasympathetic modulation in patients with chronic Chagas disease (CD). Here we investigated whether the exposure of M R to such antibodies could impair agonist-induced receptor activation, leading to the inhibition of associated signaling pathways. Preincubation of M R-expressing HEK 293T cells with serum IgG fractions from chagasic patients with cardiovascular dysautonomia, followed by the addition of carbachol, resulted in the attenuation of agonist-induced G protein activation and arrestin-2 recruitment. These effects were not mimicked by the corresponding Fab fractions, suggesting that they occur through receptor crosslinking. IgG autoantibodies did not enhance M R/arrestin interaction or promote M R internalization, suggesting that their inhibitory effects are not likely a result of short-term receptor regulation. Rather, these immunoglobulins could function as negative allosteric modulators of acetylcholine-mediated responses, thereby contributing to the development of parasympathetic dysfunction in patients with CD.