Gallbladder Na+ absorption is linked to gallstone formation in prairie dogs. We previously reported Na+/H+ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I sc, (11.6 ± 0.5 µA · cm−2), potential differences, V t (2.1 ± 0.2 mV), and resistance, R t (169 ± 12 Ω · cm2). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable 22Na+ uptake under a H+ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of 22Na+ uptake was 21.4 ± 1.3 nmol · mg prot−1 · min−1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs’ total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V max 9.2 ± 0.3 nmol · mg prot−1 · min−1; K m 11.4 ± 1.4 mM Na+). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na+ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.