The five muscarinic acetylcholine receptors (mAChRs M1-M5) form an important family of class A GPCRs widely expressed throughout the central and peripheral nervous systems, where they mediate many of the actions of the neurotransmitter, acetylcholine. In contrast to the M1-M4mAChRs, little is known about the M5 subtype. The M5 mAChR represents only 2% of the total mAChR population in the brain, but it is thought to play an important role in the mesolimbic reward pathway and may thus represent a target for treatment of drug addiction. Here we report the first crystal structure of the M5 mAChR bound to the high-affinity inverse agonist tiotropium at a resolution of 2.5 Å. With crystal structures for all five family members now at hand, we have the opportunity to understand the structural differences between these closely related receptors. In addition, recently discovered highly selective M5 allosteric modulators have provided the opportunity for a deeper understanding of the receptor’s physiological and therapeutic relevance; however, questions remain about how such ligands interact with the receptor. In an attempt to address these questions, we have utilized structural, biophysical, and pharmacological experiments to examine how different types of allosteric modulators bind to the M5 receptor. Overall, our results indicate that the highly selective M5 allosteric modulators do not bind to the prototypical mAChR allosteric site.