Brain cholinergic modulation is essential for learning-induced plasticity of the auditory cortex. The pedunculopontine tegmental nucleus (PPTg) is an important cholinergic nucleus in the brainstem, and appears to be involved in learning and subcortical plasticity. This study confirms the involvement of the PPTg in the plasticity of the auditory cortex in mice. We show here that electrical stimulation of the PPTg paired with a tone induced drastic changes in the frequency tunings of auditory cortical neurons. Importantly, the changes in frequency tuning were highly specific to the frequency of the paired tone; the best frequency of auditory cortical neurons shifted towards the frequency of the paired tone. We further demonstrated that such frequency-specific plasticity was largely eliminated by either thalamic or cortical application of the muscarinic acetylcholine receptor antagonist atropine. Our finding suggests that the PPTg significantly contributes to auditory cortical plasticity via the auditory thalamus and cholinergic basal forebrain.