Organic anion transporter 10 (OAT10, SLC22A13) is abundantly expressed in renal tubules and mediates the transport of organic anions, including nicotinate, β-hydroxybutyrate, p-aminohippurate, and orotate. In vitro transport assays using Xenopus oocytes and HEK293 cells revealed that the apparent substrate selectivity of OAT10 is distinct between the two expression systems, with particularly lower uptake of β-hydroxybutyrate in HEK293 cells. By means of co-immunoprecipitation followed by LC-MS/MS-based proteomic analysis, we found monocarboxylate transporter 1 (MCT1, SLC16A1) as an endogenous transporter physically interacting with OAT10 in HEK293 cells. The uptake of β-hydroxybutyrate and nicotinate, common substrates of OAT10 and MCT1, was increased by the knockdown of MCT1 in OAT10-expressing HEK293 cells, whereas the uptake of orotate, a substrate only for OAT10, was unaffected. These results suggest that MCT1 mediates the efflux of β-hydroxybutyrate and nicotinate that were taken up by adjacently located OAT10 in HEK293 cells, resulting in distinct apparent substrate selectivity of OAT10 from that in Xenopus oocytes. Our findings provide a general warning that unexpected interactions with endogenous transporters in specific expression systems may interfere with assessing the properties of transporters.