URAT1 is responsible for renal reabsorption of uric acid (UA) and is a target of uricosuric agents. Newly developed uricosuric agent dotinurad selectively inhibits URAT1. Interestingly, in vitro experimentally obtained Ki of dotinurad was larger than in vivo Ki estimated by model analysis of clinical uricosuric effect (Pharmacol Res Perspect. 7(6):e00533, 2019). In the present study, we precisely analysed inhibition mechanism of URAT1 by dotinurad. Xenopus oocytes that were injected with cRNA of URAT1 were used for analysis of URAT1 activity. When dotinurad was preincubated with oocytes, UA uptake were decreased more strongly than without preincubation. Furthermore, when dotinurad was directly injected into oocytes and immediately measured UA uptake without dotinurad in the uptake medium, UA uptake was decreased, while such pre-injection effect was not observed by conventional URAT1 inhibitor benzbromarone. These results demonstrated that dotinurad specifically affects URAT1 from intracellular side (trans-inhibition) in addition to cis-inhibition. Accordingly, clinical effect of dotinurad is explained by inhibition of URAT1 from both tubular lumen side (cis-inhibition) and intracellular side (trans-inhibition) and such dual mechanism may explain apparent difference of URAT1 inhibition potency between in vitro and in vivo.