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 alpha 4 beta 2* 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-beta-erythroidine, an antagonist of alpha 4 beta 2* nAChRs, but not by alpha-bungarotoxin, an antagonist of alpha 7 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 beta 2 nAChR expression was knocked down. Finally, pre-treatment with nicotine enhanced the Ca2+-response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre-synaptic alpha 4 beta 2 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.