Mechanism-based inactivation (MBI) of cytochrome P450 (CYP) 3A by verapamil and the resulting drug-drug interactions have been studied in vitro, but the inhibition of verapamil on its own metabolic clearance in clinic, namely auto-inhibition of verapamil metabolism, has never been reproduced in vitro. This paper aimed to evaluate the utility of gel entrapped rat hepatocytes in reflecting such metabolic auto-inhibition using hepatocyte monolayer as a control. Despite being a similar concentration- and time-dependent profile, auto-inhibition of verapamil metabolism showed apparent distinctions between the two culture models. Firstly, gel entrapped hepatocytes were more sensitive to such inhibition, which could be largely due to their higher CYP3A activity detected by the formation rates of 6β-hydroxy testosterone and 1’-hydroxy midazolam. Furthermore, the inhibitory effect of ketoconazole and verapamil on CYP 3A activity as well as the reduction of verapamil intrinsic clearance (CL_int) by ketoconazole was only observed in gel-entrapped hepatocytes. In this respect, the involvement of CYP3A in auto-inhibition of verapamil metabolism could be illustrated in gel-entrapped hepatocytes but not in hepatocyte monolayer. All of these results indicated that hepatocytes of gel entrapment reflected more of verapamil metabolic auto-inhibition than hepatocyte monolayer and could serve as a suitable system for investigating drug metabolism.