Various substances called uremic toxins (UTs) accumulate in the blood of patients with chronic kidney disease (CKD) and induce unfavorable effects on the body. It has been reported that some kinds of UTs are excreted extensively in the urine via renal transporters. This characteristic of UTs often becomes a factor for influencing pharmacokinetics of drugs in CKD patients. Even now, however, information on the interactions between UTs and drugs in the process of renal excretion remains limited. Methotrexate (MTX) is widely used for the treatment of rheumatoid and leukemia. It is known that MTX is predominantly excreted in the urine and that this process is mediated by organic anion transporters (OATs). In this study, we investigated the inhibitory effects of two anionic UTs, indoxyl sulfate (IS) and indoleacetic acid (IA), as well as creatinine (Cr) on the renal transport of methotrexate (MTX) using rat renal cortical slices. IS and IA, both substrates for OATs, significantly inhibited the uptake of 50 μM MTX in a concentration-dependent manner at 0.1 mM and 1 mM. In the presence of 1 mM Cr, a cationic guanidino compound, the uptake of MTX was significantly decreased, indicating that Cr is capable of interfering with OATs. In conclusion, it was suggested that the urinary excretion of MTX is extensively suppressed through interactions via OATs when IS, IA, and Cr exist a high concentrations in the blood of CKD patients.