MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+-substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild-type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na+ ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di-Na+ binding state is likely more efficient for conformational changes of NorM_VC toward the inward-facing conformation than single-Na+ binding state. The observation of two Na+ binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na+ binding and enrich our knowledge of the transport mechanism of secondary transporters. Proteins 2014; 82:240–249. © 2013 Wiley Periodicals, Inc.