The nicotinic acetylcholine receptor (nAChR) is a ligand gated ion channel protein, composed of three domains: a transmembrane domain (TM-domain), extracellular domain (EC-domain), and intracellular domain (IC-domain). Due to its biological importance, much experimental and theoretical research has been carried out to explore its mechanisms of gating and selectivity, but there are still many unresolved issues, especially on the ion selectivity. Moreover, most of the previous theoretical work has concentrated on the TM-domain or EC-domain of nAChR, which may be insufficient to understand the entire structure–function relation. In this work, we perform molecular dynamics, Brownian dynamics simulations and continuum electrostatic calculations to investigate the role of different nAChR domains in ion conduction and selectivity. The results show that although both the EC and IC domains contain strong negative charges that create large cation concentrations at either end of the pore, this alone is not sufficient to create the observed cation selectivity and may play a greater role in determining the channel conductance. The presence of cations in the wide regions of the pore can screen out the protein charge allowing anions to enter, meaning that local regions of the TM-domain are most likely responsible for discriminating between ions. These new results complement our understanding about the ion conduction and selectivity mechanism of nAChR.