Behavioural flexibility is mediated through the neural circuitry linking the prefrontal cortex and basal ganglia. Here we conduct selective elimination of striatal cholinergic interneurons in transgenic rats by immunotoxin-mediated cell targeting. Elimination of cholinergic interneurons from the dorsomedial striatum (DMS), but not from the dorsolateral striatum, results in enhanced reversal and extinction learning, sparing the acquisition of place discrimination. This enhancement is prevented by infusion of a non-selective muscarinic acetylcholine receptor agonist into the DMS either in the acquisition, reversal or extinction phase. In addition, gene-specific silencing of M-4 muscarinic receptor by lentiviral expression of short hairpin RNA (shRNA) mimics the place reversal learning promoted by cholinergic elimination, whereas shRNA-mediated gene silencing of M-1 muscarinic receptor shows the normal performance of reversal learning. Our data indicate that DMS cholinergic interneurons inhibit behavioural flexibility, mainly through the M-4 muscarinic receptor, suggesting that this role is engaged to the stabilization of acquired reward contingency and the suppression of response switch to changed contingency.