Sleep regulation involves interdependent signaling among specialized neurons in distributed brain regions. Although acetylcholine promotes wakefulness and rapid eye movement (REM) sleep, it is unclear whether the cholinergic pathway is essential (i.e., absolutely required) for REM sleep because of redundancy from neural circuits to molecules. First, we demonstrate that synaptic inhibition of TrkA+ cholinergic neurons causes a severe short-sleep phenotype and that sleep reduction is mostly attributable to a shortened sleep duration in the dark phase. Subsequent comprehensive knockout of acetylcholine receptor genes by the triple-target CRISPR method reveals that a similar short-sleep phenotype appears in the knockout of two Gq-type acetylcholine receptors Chrm1 and Chrm3. Strikingly, Chrm1 and Chrm3 double knockout chronically diminishes REM sleep to an almost undetectable level. These results suggest that muscarinic acetylcholine receptors, Chrm1 and Chrm3, are essential for REM sleep.