Apical broad-spectrum neutral (0) amino acid transporter B0AT1 (SLC6A19) is mainly expressed in small intestine and kidney, mediating the transport of all neutral amino acids in a Na+-dependent manner. Its ancillary proteins, carboxypeptidase angiotensin-converting enzyme 2 (ACE2) and the homolog collectrin (TMEM27, transmembrane protein 27), are critical for the surface expression and catalytic transport function in small intestine and kidney, respectively. B0AT1 nullizygous mice have improved insulin sensitivity and show reduced weight gain on a HFD (high fat diet) compared to wild-type mice. Hence, pharmacological inhibition of B0AT1 using chemical compounds could lead to new drugs to treat type 2 diabetes and its related metabolic disorders. In this study, I characterised a series of novel inhibitors of the B0AT1 transporter and identified two leading compounds. Benztropine (NSC63912) was identified as a competitive inhibitor showing an IC50 of 44±9µM; whereas 2-benzyl-1-(3-phenylpropyl)piperidine (NSC22789) was identified as a non-competitive inhibitor with an IC50 of 90±21µM. These two compounds were selective with regard to related transporters (system L, ASCT2), and they blocked substrate (Leucine) uptake in both Xenopus oocytes overexpressing B0AT1 and collectrin, and inverted mouse small intestine. The tools established in this study can be widely used to identify new transport inhibitors. Using these tools we were able to identify compounds that can be used to study epithelial transporters or be developed further through medicinal chemistry.