In our previous study, Rhizoma Coptidis extract was found to exert more potent inhibitory effect than its major component berberine towards urease from Helicobacter pylori (HPU) and jack bean (JBU). In continuation of our work, the present study was designed to further comparatively investigate the urease inhibitory activities of five major protoberberine alkaloids in Rhizoma Coptidis, namely berberine, palmatine, coptisine, epiberberine, jateorhizine to identify the bioactive constituent, and illuminate the potential mechanism of action. Results indicated that the five protoberberine alkaloids acted as concentration-dependent inactivators of urease with IC50 values ranging between 3.0 and 5087 μM for HPU and 2.3–> 10,000 μM for JBU, respectively. Notably, epiberberine (EB) was found to be the most potent inhibitor against both ureases with IC50 values of 3.0 ± 0.01 μM for HPU and 2.3 ± 0.01 μM for JBU, which was more effective than the standard urease inhibitor, acetohydroxamic acid (83 ± 0.01 μM for HPU and 22 ± 0.01 μM for JBU, respectively). Further kinetic analysis revealed that the type of EB inhibition against HPU was slow-binding and uncompetitive, with Ki of 10.6 ± 0.01 μM, while slow-binding and competitive against JBU with Ki of 4.6 ± 0.01 μM. Addition of thiol reagents, such as l-cysteine, glutathione and dithiothreitol, significantly abolished the inhibition, while Ni2+ competitive inhibitors, boric acid and sodium fluoride, synergetically inhibited urease with EB, indicating the obligatory role of the active site sulfhydryl group for the inhibition. In addition, binding of EB with the urease proved to be reversible, as about 65% and 90% enzymatic activity of HPU and JBU, respectively, could be restored by dithiothreitol application. These findings highlighted the potential role of Rhizoma Coptidis protoberberine alkaloids, especially EB, as a lead urease inhibitor in the treatment of diseases associated with ureolytic bacteria. Thus, EB had good potential for further development into a promising therapeutic approach for the treatment of urease-related diseases.