- The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H2 receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05–10 mM in both apical–basolateral (AP–BL) and BL–AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL–AP than AP–BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC50 of verapamil on nizatidine P-gp secretion was 1.2 × 10−2 mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J max = 5.7 × 10−3 nmol∙cm−2∙s−1 and K m = 2.2 mM) and one nonsaturable component (K d = 7 × 10−4 μL∙cm−2∙s−1). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V max and K m estimated for nizatidine P-gp-mediated secretion were 4 × 10−3 nmol∙cm−2∙s−1 and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug–drug interactions.