Cholinergic neurons in the CNS are involved in synaptic plasticity and cognition. Both muscarinic and nicotinic acetylcholine receptors (nAChRs) influence plasticity and cognitive function. The mechanism underlying nAChR-induced plasticity, however, has remained elusive. Here, we demonstrate morphological changes in dendritic spines following activation of α4β2* nAChRs, which are expressed on glutamatergic pre-synaptic termini of cultured hippocampal neurons. Exposure of the neurons to nicotine resulted in a lateral enlargement of spine heads. This was abolished by dihydro-β-erythroidine, an antagonist of α4β2* nAChRs, but not by α-bungarotoxin, an antagonist of α7 nAChRs. Tetanus toxin or a mixture of 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, antagonists of NMDA- and AMPA-type glutamate receptors, blocked the nicotine-induced spine remodeling. In addition, nicotine exerted full spine-enlarging response in the post-synaptic neuron whose β2 nAChR expression was knocked down. Finally, pre-treatment with nicotine enhanced the Ca(2+)-response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre-synaptic α4β2 nAChRs, resulting in the modulation of spinal architecture and responsiveness. The present findings may represent one of the cellular mechanisms underlying cholinergic tuning of brain function. Activation of nicotinic acetylcholine receptors (nAChRs) in brain influences plasticity and cognition. Here, activation of α4β2* nAChRs, which are expressed on glutamatergic presynaptic termini, results in the enlargement of dendritic spines through the modulation of the glutamatergic neurotransmission. The remodeled spinal architecture might be responsible for the change in responsiveness of neural circuitry, leading to cholinergic tuning of brain function.