The effect of pharmaceutical compounds on the rat kidney B0AT1 transporter in proteoliposomes has been screened. To this aim, inhibition of the transport activity by the different compounds was measured on Na+-[3H]glutamine co-transport in the presence of membrane potential positive outside. Most of the tested drugs had no effect on the transport activity. Some compounds exhibited inhibitory effects from 5 to 88% at concentration of 300 μM. Among the tested compounds, only the anti-inflammatory drug nimesulide exerted potent inhibition on B0AT1. From dose response analysis, an IC50 value of 23 μM was found. Inhibition kinetic analysis was performed: noncompetitive inhibition of the glutamine transport was observed while competitive behaviour was found when the inhibition was analyzed with respect to the Na+ concentration. Several molecules harbouring functional groups of nimesulide (analogues) were tested as inhibitors. None among the tested molecules has the capacity to inhibit the transport with the exception of the compound NS-398, whose chemical structure is very close to that of whole nimesulide. The IC50 for this compound was 131 μM. Inhibition kinetics showed behaviour of NS-398 identical to that of nimesulide, i.e., noncompetitive inhibition respect to glutamine and competitive inhibition respect to Na+. Molecular docking of nimesulide suggested that this drug is able to bind B0AT1 in an external dedicated binding site and that its binding produces a steric hindrance effect of the protein translocation path abolishing the transporter activity.