Anion channels are an essential component of the cells for keeping them alive and mediating their diverse functions. Although many anions can permeate anion channels, chloride and bicarbonate are the two most abundant anions that can be the charge carrier of anion channels in animal cells. Increasing evidence indicates that bicarbonate permeation though anion channel is involved in many basic biologic processes ranging from epithelial fluid secretion to neuronal excitation. Interestingly, ion selectivity of anion channels is not fixed and can be dynamically altered under specific cellular conditions. For example, the N-terminal region of WNK1 increases the bicarbonate permeability (PHCO3/PCl) of CFTR anion channel via a [Cl−]i-dependent physical association and that defects in this process predispose to CFTR mutation-related disorders. By employing an integrated study of combined molecular, physiological, structural, and mathematical approaches, we provide evidence that electric permittivity and channel pore diameter are cardinal features, which determine the ion selectivity of anion channels. Pore size change affects the bicarbonate permeability of anion channels by altering energy barriers of size-exclusion and ion dehydration of bicarbonate permeation. These findings provide key insights into the mechanism of how the ion permeation and selectivity of anion channels are determined.