DREADDs have revolutionized neuroscience research and hold promise for therapeutic applications. While their ability to modulate neuronal activity is well established, the specific signaling pathways they can trigger remain only partially explored. In this project, we therefore examine the signaling preferences of the human muscarinic acetylcholine receptor M3 and M4 (hM3/4) and the respective DREADDs (hM3D and hM4D). Additionally, we evaluate the impact of the individual single point mutations Y3.33C and A5.46G, which convert the wild type receptors to the DREADD. We investigated signaling of all receptors to 13 different heterotrimeric G-protein complexes and β-arrestin, using the bioluminescence resonance energy transfer based TRUPATH and luminescence based Presto-TANGO assays. In this study, we identified substantial changes in signaling between hM3/4 and the associated hM3D/4D DREADDs. hM3D selectively couples Gαq/11 while hM3 couples to multiple subunit classes. hM4D gains Gα13/Q signaling and showed significantly reduced efficacy in Gαi activation in comparison to hM4. Additionally, we observed increased basal hM3D/4D β-arrestin signaling compared to hM3/4. Investigating the impact of the single point mutations in hM3/4 further allowed us to pinpoint the modifications responsible for the observed signaling alterations. The activation of different G-protein subunit families leads to specific downstream signaling effects, and, subsequently, to a wide variety of physiological responses. A detailed characterization of DREADDs signaling is therefore pivotal for improved understanding of DREADD overexpression and activation dependent behavioral modifications, and essential for potential translational applications.