The accessory olfactory bulb (AOB), the first relay of chemosensory information in the Vomeronasal system, receives extensive cholinergic innervation from the basal forebrain. Cholinergic modulation of neuronal activity in the olfactory bulb has been hypothesized to play an important role in olfactory processing; however, little is known about the cellular actions of acetylcholine (ACh) within the AOB. Here using in vitro slice preparation, we show that muscarinic acetylcholine receptor (mAChR) activation increases neuronal excitability of granule and mitral/tufted cells (GCs and MCs) in the AOB. Activation of mAChRs increased excitability of GCs by three distinct mechanisms: induction of a long-lasting depolarization, activation of a slow afterdepolarization (sADP), and an increase in excitatory glutamatergic input due to MC depolarization. The depolarization and sADP were elicited by the selective agonist 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1-aminium chloride (100 μM) and blocked by low concentrations of pirenzepine (300 nM), indicating that they result from activation of M1-like mAChRs. In contrast, cholinergic stimulation increased the excitability of MCs via recruitment of nicotinic AChRs (nAChRs) and M1-like mAChRs. Submaximal activation of these receptors, however, decreased the excitability of MCs. Surprisingly, we found that unlike GCs in the main olfactory bulb, GCs in the AOB are excited by mAChR activation in young postnatal neurons, suggesting marked differences in cholinergic regulation of development between these two regions of the olfactory bulb.